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1998 |
Shortle, D; Simons, K T; Baker, David Clustering of low-energy conformations near the native structures of small proteins Journal Article Proceedings of the National Academy of Sciences of the United States of America, 95 , pp. 11158-62, 1998, ISSN: 0027-8424. @article{213, title = {Clustering of low-energy conformations near the native structures of small proteins}, author = { D Shortle and K T Simons and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/shortle98A.pdf}, issn = {0027-8424}, year = {1998}, date = {1998-09-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {95}, pages = {11158-62}, abstract = {Recent experimental studies of the denatured state and theoretical analyses of the folding landscape suggest that there are a large multiplicity of low-energy, partially folded conformations near the native state. In this report, we describe a strategy for predicting protein structure based on the working hypothesis that there are a greater number of low-energy conformations surrounding the correct fold than there are surrounding low-energy incorrect folds. To test this idea, 12 ensembles of 500 to 1,000 low-energy structures for 10 small proteins were analyzed by calculating the rms deviation of the Calpha coordinates between each conformation and every other conformation in the ensemble. In all 12 cases, the conformation with the greatest number of conformations within 4-A rms deviation was closer to the native structure than were the majority of conformations in the ensemble, and in most cases it was among the closest 1 to 5%. These results suggest that, to fold efficiently and retain robustness to changes in amino acid sequence, proteins may have evolved a native structure situated within a broad basin of low-energy conformations, a feature which could facilitate the prediction of protein structure at low resolution.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Recent experimental studies of the denatured state and theoretical analyses of the folding landscape suggest that there are a large multiplicity of low-energy, partially folded conformations near the native state. In this report, we describe a strategy for predicting protein structure based on the working hypothesis that there are a greater number of low-energy conformations surrounding the correct fold than there are surrounding low-energy incorrect folds. To test this idea, 12 ensembles of 500 to 1,000 low-energy structures for 10 small proteins were analyzed by calculating the rms deviation of the Calpha coordinates between each conformation and every other conformation in the ensemble. In all 12 cases, the conformation with the greatest number of conformations within 4-A rms deviation was closer to the native structure than were the majority of conformations in the ensemble, and in most cases it was among the closest 1 to 5%. These results suggest that, to fold efficiently and retain robustness to changes in amino acid sequence, proteins may have evolved a native structure situated within a broad basin of low-energy conformations, a feature which could facilitate the prediction of protein structure at low resolution. |
Bystroff, C; Baker, D Prediction of local structure in proteins using a library of sequence-structure motifs Journal Article Journal of molecular biology, 281 , pp. 565-77, 1998, ISSN: 0022-2836. @article{311, title = {Prediction of local structure in proteins using a library of sequence-structure motifs}, author = { C Bystroff and D Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/bystroff98A.pdf}, issn = {0022-2836}, year = {1998}, date = {1998-08-01}, journal = {Journal of molecular biology}, volume = {281}, pages = {565-77}, abstract = {We describe a new method for local protein structure prediction based on a library of short sequence pattern that correlate strongly with protein three-dimensional structural elements. The library was generated using an automated method for finding correlations between protein sequence and local structure, and contains most previously described local sequence-structure correlations as well as new relationships, including a diverging type-II beta-turn, a frayed helix, and a proline-terminated helix. The query sequence is scanned for segments 7 to 19 residues in length that strongly match one of the 82 patterns in the library. Matching segments are assigned the three-dimensional structure characteristic of the corresponding sequence pattern, and backbone torsion angles for the entire query sequence are then predicted by piecing together mutually compatible segment predictions. In predictions of local structure in a test set of 55 proteins, about 50% of all residues, and 76% of residues covered by high-confidence predictions, were found in eight-residue segments within 1.4 A of their true structures. The predictions are complementary to traditional secondary structure predictions because they are considerably more specific in turn regions, and may contribute to ab initio tertiary structure prediction and fold recognition.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe a new method for local protein structure prediction based on a library of short sequence pattern that correlate strongly with protein three-dimensional structural elements. The library was generated using an automated method for finding correlations between protein sequence and local structure, and contains most previously described local sequence-structure correlations as well as new relationships, including a diverging type-II beta-turn, a frayed helix, and a proline-terminated helix. The query sequence is scanned for segments 7 to 19 residues in length that strongly match one of the 82 patterns in the library. Matching segments are assigned the three-dimensional structure characteristic of the corresponding sequence pattern, and backbone torsion angles for the entire query sequence are then predicted by piecing together mutually compatible segment predictions. In predictions of local structure in a test set of 55 proteins, about 50% of all residues, and 76% of residues covered by high-confidence predictions, were found in eight-residue segments within 1.4 A of their true structures. The predictions are complementary to traditional secondary structure predictions because they are considerably more specific in turn regions, and may contribute to ab initio tertiary structure prediction and fold recognition. |
Grantcharova, V P; Riddle, D S; Santiago, J V; Baker, David Important role of hydrogen bonds in the structurally polarized transition state for folding of the src SH3 domain Journal Article Nature structural biology, 5 , pp. 714-20, 1998, ISSN: 1072-8368. @article{204, title = {Important role of hydrogen bonds in the structurally polarized transition state for folding of the src SH3 domain}, author = { V P Grantcharova and D S Riddle and J V Santiago and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/grantcharova98A.pdf}, issn = {1072-8368}, year = {1998}, date = {1998-08-01}, journal = {Nature structural biology}, volume = {5}, pages = {714-20}, abstract = {Experimental and theoretical studies on the folding of small proteins such as the chymotrypsin inhibitor 2 (CI-2) and the P22 Arc repressor suggest that the folding transition state is an expanded version of the native state with most interactions partially formed. Here we report that this picture does not hold generally: a hydrogen bond network involving two beta-turns and an adjacent hydrophobic cluster appear to be formed in the folding transition state of the src SH3 domain, while the remainder of the polypeptide chain is largely unstructured. Comparison with data on other small proteins suggests that this structural polarization is a consequence of the topology of the SH3 domain fold. The non-uniform distribution of structure in the folding transition state provides a challenging test for computational models of the folding process.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Experimental and theoretical studies on the folding of small proteins such as the chymotrypsin inhibitor 2 (CI-2) and the P22 Arc repressor suggest that the folding transition state is an expanded version of the native state with most interactions partially formed. Here we report that this picture does not hold generally: a hydrogen bond network involving two beta-turns and an adjacent hydrophobic cluster appear to be formed in the folding transition state of the src SH3 domain, while the remainder of the polypeptide chain is largely unstructured. Comparison with data on other small proteins suggests that this structural polarization is a consequence of the topology of the SH3 domain fold. The non-uniform distribution of structure in the folding transition state provides a challenging test for computational models of the folding process. |
Kim, D E; Gu, H; Baker, D The sequences of small proteins are not extensively optimized for rapid folding by natural selection Journal Article Proceedings of the National Academy of Sciences of the United States of America, 95 , pp. 4982-6, 1998, ISSN: 0027-8424. @article{312, title = {The sequences of small proteins are not extensively optimized for rapid folding by natural selection}, author = { D E Kim and H Gu and D Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/kim98A.pdf}, issn = {0027-8424}, year = {1998}, date = {1998-04-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {95}, pages = {4982-6}, abstract = {The thermodynamic stabilities of small protein domains are clearly subject to natural selection, but it is less clear whether the rapid folding rates typically observed for such proteins are consequences of direct evolutionary optimization or reflect intrinsic physical properties of the polypeptide chain. This issue can be investigated by comparing the folding rates of laboratory-generated protein sequences to those of naturally occurring sequences provided that the method by which the sequences are generated has no kinetic bias. Herein we report the folding thermodynamics and kinetics of 12 heavily mutated variants of the small IgG binding domain of protein L retrieved from high-complexity combinatorial libraries by using a phage-display selection for proper folding that does not discriminate between rapidly and slowly folding proteins. Although the stabilities of all variants were decreased, many of the variants fold faster than wild type. Taken together with similar results for the src homology 3 domain, this observation suggests that the sequences of small proteins have not been extensively optimized for rapid folding; instead, rapid folding appears to be a consequence of selection for stability.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The thermodynamic stabilities of small protein domains are clearly subject to natural selection, but it is less clear whether the rapid folding rates typically observed for such proteins are consequences of direct evolutionary optimization or reflect intrinsic physical properties of the polypeptide chain. This issue can be investigated by comparing the folding rates of laboratory-generated protein sequences to those of naturally occurring sequences provided that the method by which the sequences are generated has no kinetic bias. Herein we report the folding thermodynamics and kinetics of 12 heavily mutated variants of the small IgG binding domain of protein L retrieved from high-complexity combinatorial libraries by using a phage-display selection for proper folding that does not discriminate between rapidly and slowly folding proteins. Although the stabilities of all variants were decreased, many of the variants fold faster than wild type. Taken together with similar results for the src homology 3 domain, this observation suggests that the sequences of small proteins have not been extensively optimized for rapid folding; instead, rapid folding appears to be a consequence of selection for stability. |
Rank, J A; Baker, David Contributions of solvent-solvent hydrogen bonding and van der Waals interactions to the attraction between methane molecules in water Journal Article Biophysical chemistry, 71 , pp. 199-204, 1998, ISSN: 0301-4622. @article{210, title = {Contributions of solvent-solvent hydrogen bonding and van der Waals interactions to the attraction between methane molecules in water}, author = { J A Rank and David Baker}, issn = {0301-4622}, year = {1998}, date = {1998-04-01}, journal = {Biophysical chemistry}, volume = {71}, pages = {199-204}, abstract = {The contribution of solvent-solvent hydrogen bonding and van der Waals interactions to the attraction between methane molecules in water was investigated by comparing the potential of mean force (PMF) between two methane molecules in TIP4P water to those in a series of related liquids in which the solvent-solvent interactions were progressively turned off while keeping the solvent-solute interactions unchanged. The magnitude of the attraction between methanes was not significantly changed when the hydrogen bonding interaction between solvent molecules was eliminated and the solvent was maintained in the liquid state by increasing either the pressure or the magnitude of the solvent-solvent van der Waals interaction. However, when solvent-solvent excluded volume interactions were eliminated, the methane molecules interacted no more strongly than in the gas phase. The results are consistent with the idea that the primary contribution of hydrogen bonding to the hydrophobic interaction is to keep water molecules in a liquid state; at constant density, packing interactions rather than hydrogen bonding appear to be critical as suggested by scaled particle theories of solvation. The overall shape of the PMF was, however, changed in the absence of hydrogen bonding, pointing to an influence of hydrogen bonding on the detailed form of the interactions between nonpolar solutes in water. The effects of correlations between the configurations sampled during the Monte Carlo procedure used in the free energy calculations on the estimation of errors was also characterized.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The contribution of solvent-solvent hydrogen bonding and van der Waals interactions to the attraction between methane molecules in water was investigated by comparing the potential of mean force (PMF) between two methane molecules in TIP4P water to those in a series of related liquids in which the solvent-solvent interactions were progressively turned off while keeping the solvent-solute interactions unchanged. The magnitude of the attraction between methanes was not significantly changed when the hydrogen bonding interaction between solvent molecules was eliminated and the solvent was maintained in the liquid state by increasing either the pressure or the magnitude of the solvent-solvent van der Waals interaction. However, when solvent-solvent excluded volume interactions were eliminated, the methane molecules interacted no more strongly than in the gas phase. The results are consistent with the idea that the primary contribution of hydrogen bonding to the hydrophobic interaction is to keep water molecules in a liquid state; at constant density, packing interactions rather than hydrogen bonding appear to be critical as suggested by scaled particle theories of solvation. The overall shape of the PMF was, however, changed in the absence of hydrogen bonding, pointing to an influence of hydrogen bonding on the detailed form of the interactions between nonpolar solutes in water. The effects of correlations between the configurations sampled during the Monte Carlo procedure used in the free energy calculations on the estimation of errors was also characterized. |
Plaxco, K W; Simons, K T; Baker, David Contact order, transition state placement and the refolding rates of single domain proteins Journal Article Journal of molecular biology, 277 , pp. 985-94, 1998, ISSN: 0022-2836. @article{207, title = {Contact order, transition state placement and the refolding rates of single domain proteins}, author = { K W Plaxco and K T Simons and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/plaxco98B.pdf}, issn = {0022-2836}, year = {1998}, date = {1998-04-01}, journal = {Journal of molecular biology}, volume = {277}, pages = {985-94}, abstract = {Theoretical studies have suggested relationships between the size, stability and topology of a protein fold and the rate and mechanisms by which it is achieved. The recent characterization of the refolding of a number of simple, single domain proteins has provided a means of testing these assertions. Our investigations have revealed statistically significant correlations between the average sequence separation between contacting residues in the native state and the rate and transition state placement of folding for a non-homologous set of simple, single domain proteins. These indicate that proteins featuring primarily sequence-local contacts tend to fold more rapidly and exhibit less compact folding transition states than those characterized by more non-local interactions. No significant relationship is apparent between protein length and folding rates, but a weak correlation is observed between length and the fraction of solvent-exposed surface area buried in the transition state. Anticipated strong relationships between equilibrium folding free energy and folding kinetics, or between chemical denaturant and temperature dependence-derived measures of transition state placement, are not apparent. The observed correlations are consistent with a model of protein folding in which the size and stability of the polypeptide segments organized in the transition state are largely independent of protein length, but are related to the topological complexity of the native state. The correlation between topological complexity and folding rates may reflect chain entropy contributions to the folding barrier.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Theoretical studies have suggested relationships between the size, stability and topology of a protein fold and the rate and mechanisms by which it is achieved. The recent characterization of the refolding of a number of simple, single domain proteins has provided a means of testing these assertions. Our investigations have revealed statistically significant correlations between the average sequence separation between contacting residues in the native state and the rate and transition state placement of folding for a non-homologous set of simple, single domain proteins. These indicate that proteins featuring primarily sequence-local contacts tend to fold more rapidly and exhibit less compact folding transition states than those characterized by more non-local interactions. No significant relationship is apparent between protein length and folding rates, but a weak correlation is observed between length and the fraction of solvent-exposed surface area buried in the transition state. Anticipated strong relationships between equilibrium folding free energy and folding kinetics, or between chemical denaturant and temperature dependence-derived measures of transition state placement, are not apparent. The observed correlations are consistent with a model of protein folding in which the size and stability of the polypeptide segments organized in the transition state are largely independent of protein length, but are related to the topological complexity of the native state. The correlation between topological complexity and folding rates may reflect chain entropy contributions to the folding barrier. |
Plaxco, K W; Riddle, D S; Grantcharova, V; Baker, David Simplified proteins: minimalist solutions to the protein folding problem Journal Article Current opinion in structural biology, 8 , pp. 80-5, 1998, ISSN: 0959-440X. @article{208, title = {Simplified proteins: minimalist solutions to the protein folding problem}, author = { K W Plaxco and D S Riddle and V Grantcharova and David Baker}, issn = {0959-440X}, year = {1998}, date = {1998-02-01}, journal = {Current opinion in structural biology}, volume = {8}, pages = {80-5}, abstract = {Recent research has suggested that stable, native proteins may be encoded by simple sequences of fewer than the full set of 20 proteogenic amino acids. Studies of the ability of simple amino acid sequences to encode stable, topologically complex, native conformations and to fold to these conformations in a biologically relevant time frame have provided insights into the sequence determinants of protein structure and folding kinetics. They may also have important implications for protein design and for theories of the origins of protein synthesis itself.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Recent research has suggested that stable, native proteins may be encoded by simple sequences of fewer than the full set of 20 proteogenic amino acids. Studies of the ability of simple amino acid sequences to encode stable, topologically complex, native conformations and to fold to these conformations in a biologically relevant time frame have provided insights into the sequence determinants of protein structure and folding kinetics. They may also have important implications for protein design and for theories of the origins of protein synthesis itself. |
Yi, Q; Bystroff, C; Rajagopal, P; Klevit, R E; Baker, David Prediction and structural characterization of an independently folding substructure in the src SH3 domain Journal Article Journal of molecular biology, 283 , pp. 293-300, 1998, ISSN: 0022-2836. @article{212, title = {Prediction and structural characterization of an independently folding substructure in the src SH3 domain}, author = { Q Yi and C Bystroff and P Rajagopal and R E Klevit and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/yi98A.pdf}, issn = {0022-2836}, year = {1998}, date = {1998-00-01}, journal = {Journal of molecular biology}, volume = {283}, pages = {293-300}, abstract = {Previous studies of the conformations of peptides spanning the length of the alpha-spectrin SH3 domain suggested that SH3 domains lack independently folding substructures. Using a local structure prediction method based on the I-sites library of sequence-structure motifs, we identified a seven residue peptide in the src SH3 domain predicted to adopt a native-like structure, a type II beta-turn bridging unpaired beta-strands, that was not contained intact in any of the SH3 domain peptides studied earlier. NMR characterization confirmed that the isolated peptide, FKKGERL, adopts a structure similar to that adopted in the native protein: the NOE and 3JNHalpha coupling constant patterns were indicative of a type II beta-turn, and NOEs between the Phe and the Leu side-chains suggest that they are juxtaposed as in the prediction and the native structure. These results support the idea that high-confidence I-sites predictions identify protein segments that are likely to form native-like structures early in folding.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Previous studies of the conformations of peptides spanning the length of the alpha-spectrin SH3 domain suggested that SH3 domains lack independently folding substructures. Using a local structure prediction method based on the I-sites library of sequence-structure motifs, we identified a seven residue peptide in the src SH3 domain predicted to adopt a native-like structure, a type II beta-turn bridging unpaired beta-strands, that was not contained intact in any of the SH3 domain peptides studied earlier. NMR characterization confirmed that the isolated peptide, FKKGERL, adopts a structure similar to that adopted in the native protein: the NOE and 3JNHalpha coupling constant patterns were indicative of a type II beta-turn, and NOEs between the Phe and the Leu side-chains suggest that they are juxtaposed as in the prediction and the native structure. These results support the idea that high-confidence I-sites predictions identify protein segments that are likely to form native-like structures early in folding. |
1997 |
Grantcharova, V P; Baker, David Folding dynamics of the src SH3 domain Journal Article Biochemistry, 36 , pp. 15685-92, 1997, ISSN: 0006-2960. @article{32, title = {Folding dynamics of the src SH3 domain}, author = { V P Grantcharova and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/grantcharova97A.pdf}, issn = {0006-2960}, year = {1997}, date = {1997-12-01}, journal = {Biochemistry}, volume = {36}, pages = {15685-92}, abstract = {The thermodynamics and kinetics of folding of the chicken src SH3 domain were characterized using equilibrium and stopped-flow fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) hydrogen exchange experiments. As found for other SH3 domains, guanidinium chloride (GdmCl) denaturation melts followed by both fluorescence and circular dichroism were nearly superimposable, indicating the concerted formation of secondary and tertiary structure. Kinetic studies confirmed the two-state character of the folding reaction. Except for a very slow refolding phase due to proline isomerization, both folding and unfolding traces fit well to single exponentials over a wide range of GdmCl concentrations, and no burst phase in amplitude was observed during the dead time of the stopped-flow instrument. The entropy, enthalpy, and heat capacity changes upon unfolding were determined by global fitting of temperature melts at varying GdmCl concentrations (0.4-3.7 M). Estimates of the free energy of unfolding, DeltaGUH2O, from guanidine denaturation, thermal denaturation, and kinetic experiments were in good agreement. To complement these data on the global characteristics of src SH3 folding, individual hydrogen-deuterium (HD) exchange rates were measured for approximately half of the backbone amides in 0 and 0.7 M GdmCl. The calculated free energies of the opening reaction leading to exchange (DeltaGHD) indicated that unfolding is highly cooperative--slowly exchanging protons were distributed throughout the core of the protein. The slowly exchanging protons exhibited DeltaGHD values higher than the global DeltaGUH2O by approximately 1 kcal/mol, suggesting that the denatured state might be somewhat compact under native conditions. Comparison of the src SH3 with homologous SH3 domains as well as with other small well-characterized beta-sheet proteins provides insights into the determinants of folding kinetics and protein stability.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The thermodynamics and kinetics of folding of the chicken src SH3 domain were characterized using equilibrium and stopped-flow fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) hydrogen exchange experiments. As found for other SH3 domains, guanidinium chloride (GdmCl) denaturation melts followed by both fluorescence and circular dichroism were nearly superimposable, indicating the concerted formation of secondary and tertiary structure. Kinetic studies confirmed the two-state character of the folding reaction. Except for a very slow refolding phase due to proline isomerization, both folding and unfolding traces fit well to single exponentials over a wide range of GdmCl concentrations, and no burst phase in amplitude was observed during the dead time of the stopped-flow instrument. The entropy, enthalpy, and heat capacity changes upon unfolding were determined by global fitting of temperature melts at varying GdmCl concentrations (0.4-3.7 M). Estimates of the free energy of unfolding, DeltaGUH2O, from guanidine denaturation, thermal denaturation, and kinetic experiments were in good agreement. To complement these data on the global characteristics of src SH3 folding, individual hydrogen-deuterium (HD) exchange rates were measured for approximately half of the backbone amides in 0 and 0.7 M GdmCl. The calculated free energies of the opening reaction leading to exchange (DeltaGHD) indicated that unfolding is highly cooperative--slowly exchanging protons were distributed throughout the core of the protein. The slowly exchanging protons exhibited DeltaGHD values higher than the global DeltaGUH2O by approximately 1 kcal/mol, suggesting that the denatured state might be somewhat compact under native conditions. Comparison of the src SH3 with homologous SH3 domains as well as with other small well-characterized beta-sheet proteins provides insights into the determinants of folding kinetics and protein stability. |
Gu, H; Kim, David E; Baker, David Contrasting roles for symmetrically disposed beta-turns in the folding of a small protein Journal Article Journal of molecular biology, 274 , pp. 588-96, 1997, ISSN: 0022-2836. @article{33, title = {Contrasting roles for symmetrically disposed beta-turns in the folding of a small protein}, author = { H Gu and David E Kim and David Baker}, issn = {0022-2836}, year = {1997}, date = {1997-12-01}, journal = {Journal of molecular biology}, volume = {274}, pages = {588-96}, abstract = {To investigate the role of turns in protein folding, we have characterized the effects of combinatorial and site-directed mutations in the two beta-turns of peptostreptococcal protein L on folding thermodynamics and kinetics. Sequences of folded variants recovered from combinatorial libraries using a phase display selection method were considerably more variable in the second turn than in the first turn. These combinatorial mutants as well as strategically placed point mutants in the two turns had a similar range of thermodynamic stabilities, but strikingly different folding kinetics. A glycine to alanine substitution in the second beta-turn increased the rate of unfolding more than tenfold but had little effect on the rate of folding, while mutation of a symmetrically disposed glycine residue in the first turn had little effect on unfolding but slowed the rate of folding nearly tenfold. These results demonstrate that the role of beta-turns in protein folding is strongly context-dependent, and suggests that the first turn is formed and the second turn disrupted in the folding transition state.}, keywords = {}, pubstate = {published}, tppubtype = {article} } To investigate the role of turns in protein folding, we have characterized the effects of combinatorial and site-directed mutations in the two beta-turns of peptostreptococcal protein L on folding thermodynamics and kinetics. Sequences of folded variants recovered from combinatorial libraries using a phase display selection method were considerably more variable in the second turn than in the first turn. These combinatorial mutants as well as strategically placed point mutants in the two turns had a similar range of thermodynamic stabilities, but strikingly different folding kinetics. A glycine to alanine substitution in the second beta-turn increased the rate of unfolding more than tenfold but had little effect on the rate of folding, while mutation of a symmetrically disposed glycine residue in the first turn had little effect on unfolding but slowed the rate of folding nearly tenfold. These results demonstrate that the role of beta-turns in protein folding is strongly context-dependent, and suggests that the first turn is formed and the second turn disrupted in the folding transition state. |
Doyle, R; Simons, K; Qian, H; Baker, David Local interactions and the optimization of protein folding Journal Article Proteins, 29 , pp. 282-91, 1997, ISSN: 0887-3585. @article{31, title = {Local interactions and the optimization of protein folding}, author = { R Doyle and K Simons and H Qian and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/doyle97A.pdf}, issn = {0887-3585}, year = {1997}, date = {1997-11-01}, journal = {Proteins}, volume = {29}, pages = {282-91}, abstract = {The role of local interactions in protein folding has recently been the subject of some controversy. Here we investigate an extension of Zwanzigtextquoterights simple and general model of folding in which local and nonlocal interactions are represented by functions of single and multiple conformational degrees of freedom, respectively. The kinetics and thermodynamics of folding are studied for a series of energy functions in which the energy of the native structure is fixed, but the relative contributions of local and nonlocal interactions to this energy are varied over a broad range. For funnel shaped energy landscapes, we find that 1) the rate of folding increases, but the stability of the folded state decreases, as the contribution of local interactions to the energy of the native structure increases, and 2) the amount of native structure in the unfolded state and the transition state vary considerably with the local interaction strength. Simple exponential kinetics and a well-defined free energy barrier separating folded and unfolded states are observed when nonlocal interactions make an appreciable contribution to the energy of the native structure; in such cases a transition state theory type approximation yields reasonably accurate estimates of the folding rate. Bumps in the folding funnel near the native state, which could result from desolvation effects, side chain freezing, or the breaking of nonnative contacts, significantly alter the dependence of the folding rate on the local interaction strength: the rate of folding decreases when the local interaction strength is increased beyond a certain point. A survey of the distribution of strong contacts in the protein structure database suggests that evolutionary optimization has involved both kinetics and thermodynamics: strong contacts are enriched at both very short and very long sequence separations.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The role of local interactions in protein folding has recently been the subject of some controversy. Here we investigate an extension of Zwanzigtextquoterights simple and general model of folding in which local and nonlocal interactions are represented by functions of single and multiple conformational degrees of freedom, respectively. The kinetics and thermodynamics of folding are studied for a series of energy functions in which the energy of the native structure is fixed, but the relative contributions of local and nonlocal interactions to this energy are varied over a broad range. For funnel shaped energy landscapes, we find that 1) the rate of folding increases, but the stability of the folded state decreases, as the contribution of local interactions to the energy of the native structure increases, and 2) the amount of native structure in the unfolded state and the transition state vary considerably with the local interaction strength. Simple exponential kinetics and a well-defined free energy barrier separating folded and unfolded states are observed when nonlocal interactions make an appreciable contribution to the energy of the native structure; in such cases a transition state theory type approximation yields reasonably accurate estimates of the folding rate. Bumps in the folding funnel near the native state, which could result from desolvation effects, side chain freezing, or the breaking of nonnative contacts, significantly alter the dependence of the folding rate on the local interaction strength: the rate of folding decreases when the local interaction strength is increased beyond a certain point. A survey of the distribution of strong contacts in the protein structure database suggests that evolutionary optimization has involved both kinetics and thermodynamics: strong contacts are enriched at both very short and very long sequence separations. |
Riddle, D S; Santiago, J V; Bray-Hall, S T; Doshi, N; Grantcharova, V P; Yi, Q; Baker, David Functional rapidly folding proteins from simplified amino acid sequences Journal Article Nature structural biology, 4 , pp. 805-9, 1997, ISSN: 1072-8368. @article{26, title = {Functional rapidly folding proteins from simplified amino acid sequences}, author = { D S Riddle and J V Santiago and S T Bray-Hall and N Doshi and V P Grantcharova and Q Yi and David Baker}, issn = {1072-8368}, year = {1997}, date = {1997-10-01}, journal = {Nature structural biology}, volume = {4}, pages = {805-9}, abstract = {Early protein synthesis is thought to have involved a reduced amino acid alphabet. What is the minimum number of amino acids that would have been needed to encode complex protein folds similar to those found in nature today? Here we show that a small beta-sheet protein, the SH3 domain, can be largely encoded by a five letter amino acid alphabet but not by a three letter alphabet. Furthermore, despite the dramatic changes in sequence, the folding rates of the reduced alphabet proteins are very close to that of the naturally occurring SH3 domain. This finding suggests that despite the vast size of the search space, the rapid folding of biological sequences to their native states is not the result of extensive evolutionary optimization. Instead, the results support the idea that the interactions which stabilize the native state induce a funnel shape to the free energy landscape sufficient to guide the folding polypeptide chain to the proper structure.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Early protein synthesis is thought to have involved a reduced amino acid alphabet. What is the minimum number of amino acids that would have been needed to encode complex protein folds similar to those found in nature today? Here we show that a small beta-sheet protein, the SH3 domain, can be largely encoded by a five letter amino acid alphabet but not by a three letter alphabet. Furthermore, despite the dramatic changes in sequence, the folding rates of the reduced alphabet proteins are very close to that of the naturally occurring SH3 domain. This finding suggests that despite the vast size of the search space, the rapid folding of biological sequences to their native states is not the result of extensive evolutionary optimization. Instead, the results support the idea that the interactions which stabilize the native state induce a funnel shape to the free energy landscape sufficient to guide the folding polypeptide chain to the proper structure. |
Scalley, M L; Baker, David Protein folding kinetics exhibit an Arrhenius temperature dependence when corrected for the temperature dependence of protein stability Journal Article Proceedings of the National Academy of Sciences of the United States of America, 94 , pp. 10636-40, 1997, ISSN: 0027-8424. @article{29, title = {Protein folding kinetics exhibit an Arrhenius temperature dependence when corrected for the temperature dependence of protein stability}, author = { M L Scalley and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/scalley97A.pdf}, issn = {0027-8424}, year = {1997}, date = {1997-09-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {94}, pages = {10636-40}, abstract = {The anomalous temperature dependence of protein folding has received considerable attention. Here we show that the temperature dependence of the folding of protein L becomes extremely simple when the effects of temperature on protein stability are corrected for; the logarithm of the folding rate is a linear function of 1/T on constant stability contours in the temperature-denaturant plane. This convincingly demonstrates that the anomalous temperature dependence of folding derives from the temperature dependence of the interactions that stabilize proteins, rather than from the super Arrhenius temperature dependence predicted for the configurational diffusion constant on a rough energy landscape. However, because of the limited temperature range accessible to experiment, the results do not rule out models with higher order temperature dependences. The significance of the slope of the stability-corrected Arrhenius plots is discussed.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The anomalous temperature dependence of protein folding has received considerable attention. Here we show that the temperature dependence of the folding of protein L becomes extremely simple when the effects of temperature on protein stability are corrected for; the logarithm of the folding rate is a linear function of 1/T on constant stability contours in the temperature-denaturant plane. This convincingly demonstrates that the anomalous temperature dependence of folding derives from the temperature dependence of the interactions that stabilize proteins, rather than from the super Arrhenius temperature dependence predicted for the configurational diffusion constant on a rough energy landscape. However, because of the limited temperature range accessible to experiment, the results do not rule out models with higher order temperature dependences. The significance of the slope of the stability-corrected Arrhenius plots is discussed. |
Han, K F; Bystroff, C; Baker, David Three-dimensional structures and contexts associated with recurrent amino acid sequence patterns Journal Article Protein science, 6 , pp. 1587-90, 1997, ISSN: 0961-8368. @article{36, title = {Three-dimensional structures and contexts associated with recurrent amino acid sequence patterns}, author = { K F Han and C Bystroff and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/han97A.pdf}, issn = {0961-8368}, year = {1997}, date = {1997-07-01}, journal = {Protein science}, volume = {6}, pages = {1587-90}, abstract = {We have used cluster analysis to identify recurring sequence patterns that transcend protein family boundaries. A subset of these patterns occur predominantly in a single type of local structure in proteins. Here we characterize the three-dimensional structures and contexts in which these sequence patterns occur, with particular attention to the interactions responsible for their structural selectivity.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We have used cluster analysis to identify recurring sequence patterns that transcend protein family boundaries. A subset of these patterns occur predominantly in a single type of local structure in proteins. Here we characterize the three-dimensional structures and contexts in which these sequence patterns occur, with particular attention to the interactions responsible for their structural selectivity. |
Simons, K T; Kooperberg, C; Huang, E; Baker, David Assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and Bayesian scoring functions Journal Article Journal of molecular biology, 268 , pp. 209-25, 1997, ISSN: 0022-2836. @article{28, title = {Assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and Bayesian scoring functions}, author = { K T Simons and C Kooperberg and E Huang and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/simons97A.pdf}, issn = {0022-2836}, year = {1997}, date = {1997-04-01}, journal = {Journal of molecular biology}, volume = {268}, pages = {209-25}, abstract = {We explore the ability of a simple simulated annealing procedure to assemble native-like structures from fragments of unrelated protein structures with similar local sequences using Bayesian scoring functions. Environment and residue pair specific contributions to the scoring functions appear as the first two terms in a series expansion for the residue probability distributions in the protein database; the decoupling of the distance and environment dependencies of the distributions resolves the major problems with current database-derived scoring functions noted by Thomas and Dill. The simulated annealing procedure rapidly and frequently generates native-like structures for small helical proteins and better than random structures for small beta sheet containing proteins. Most of the simulated structures have native-like solvent accessibility and secondary structure patterns, and thus ensembles of these structures provide a particularly challenging set of decoys for evaluating scoring functions. We investigate the effects of multiple sequence information and different types of conformational constraints on the overall performance of the method, and the ability of a variety of recently developed scoring functions to recognize the native-like conformations in the ensembles of simulated structures.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We explore the ability of a simple simulated annealing procedure to assemble native-like structures from fragments of unrelated protein structures with similar local sequences using Bayesian scoring functions. Environment and residue pair specific contributions to the scoring functions appear as the first two terms in a series expansion for the residue probability distributions in the protein database; the decoupling of the distance and environment dependencies of the distributions resolves the major problems with current database-derived scoring functions noted by Thomas and Dill. The simulated annealing procedure rapidly and frequently generates native-like structures for small helical proteins and better than random structures for small beta sheet containing proteins. Most of the simulated structures have native-like solvent accessibility and secondary structure patterns, and thus ensembles of these structures provide a particularly challenging set of decoys for evaluating scoring functions. We investigate the effects of multiple sequence information and different types of conformational constraints on the overall performance of the method, and the ability of a variety of recently developed scoring functions to recognize the native-like conformations in the ensembles of simulated structures. |
Scalley, M L; Yi, Q; Gu, H; McCormack, A; Yates, J R; Baker, David Kinetics of folding of the IgG binding domain of peptostreptococcal protein L. Journal Article Biochemistry, 36 , pp. 3373-82, 1997, ISSN: 0006-2960. @article{34, title = {Kinetics of folding of the IgG binding domain of peptostreptococcal protein L.}, author = { M L Scalley and Q Yi and H Gu and A McCormack and J R Yates and David Baker}, issn = {0006-2960}, year = {1997}, date = {1997-03-01}, journal = {Biochemistry}, volume = {36}, pages = {3373-82}, abstract = {The kinetics of folding of a tryptophan containing mutant of the IgG binding domain of protein L were characterized using stopped-flow circular dichroism, stopped-flow fluorescence, and HD exchange coupled with high-resolution mass spectrometry. Both the thermodynamics and kinetics of folding fit well to a simple two-state model: (1) Guanidine induced equilibrium denaturation transitions measured by fluorescence and circular dichroism were virtually superimposable. (2) The kinetics of folding/unfolding were single exponential under all conditions examined, and the rate constants obtained using all probes were similar. (3) Mass spectra from pulsed HD exchange refolding experiments showed that a species with very little protection from exchange is converted to a fully protected species (the native state) at a rate very similar to that of the overall change in tryptophan fluorescence; no intervening partially protected species were observed. (4) Rate constants (in H2O) and m values for folding and unfolding determined by fitting observed relaxation rates obtained over a broad range of denaturant concentrations to a two-state model were consistent with the equilibrium parameters deltaG and m: -RT ln(k(u)/k(f))/deltaG(U)H2O = 1.02; (m(u) + m(f))/m = 1.08. In contrast to results with a number of other proteins, there was no deviation from linearity in plots of ln k(obs) versus guanidine at low guanidine concentrations, both in the presence and absence of 0.4 M Na2SO4, suggesting that significantly stabilized intermediates do not accumulate during folding. Although all of the change in fluorescence signal during folding in phosphate buffer was accounted for by the simple exponential describing the overall folding reaction, fluorescence-quenching experiments using sodium iodide revealed a small reduction in the extent of quenching of the protein within the first two milliseconds after initiation of refolding in low concentrations of guanidine, suggesting a partial collapse of the unfolded chain may occur under these conditions. Comparison with results on the structurally and functionally similar IgG binding domain of streptococcal protein G show intriguing differences in the folding of the two proteins.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The kinetics of folding of a tryptophan containing mutant of the IgG binding domain of protein L were characterized using stopped-flow circular dichroism, stopped-flow fluorescence, and HD exchange coupled with high-resolution mass spectrometry. Both the thermodynamics and kinetics of folding fit well to a simple two-state model: (1) Guanidine induced equilibrium denaturation transitions measured by fluorescence and circular dichroism were virtually superimposable. (2) The kinetics of folding/unfolding were single exponential under all conditions examined, and the rate constants obtained using all probes were similar. (3) Mass spectra from pulsed HD exchange refolding experiments showed that a species with very little protection from exchange is converted to a fully protected species (the native state) at a rate very similar to that of the overall change in tryptophan fluorescence; no intervening partially protected species were observed. (4) Rate constants (in H2O) and m values for folding and unfolding determined by fitting observed relaxation rates obtained over a broad range of denaturant concentrations to a two-state model were consistent with the equilibrium parameters deltaG and m: -RT ln(k(u)/k(f))/deltaG(U)H2O = 1.02; (m(u) + m(f))/m = 1.08. In contrast to results with a number of other proteins, there was no deviation from linearity in plots of ln k(obs) versus guanidine at low guanidine concentrations, both in the presence and absence of 0.4 M Na2SO4, suggesting that significantly stabilized intermediates do not accumulate during folding. Although all of the change in fluorescence signal during folding in phosphate buffer was accounted for by the simple exponential describing the overall folding reaction, fluorescence-quenching experiments using sodium iodide revealed a small reduction in the extent of quenching of the protein within the first two milliseconds after initiation of refolding in low concentrations of guanidine, suggesting a partial collapse of the unfolded chain may occur under these conditions. Comparison with results on the structurally and functionally similar IgG binding domain of streptococcal protein G show intriguing differences in the folding of the two proteins. |
Rank, J A; Baker, David A desolvation barrier to hydrophobic cluster formation may contribute to the rate-limiting step in protein folding Journal Article Protein science, 6 , pp. 347-54, 1997, ISSN: 0961-8368. @article{35, title = {A desolvation barrier to hydrophobic cluster formation may contribute to the rate-limiting step in protein folding}, author = { J A Rank and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/rank97A.pdf}, issn = {0961-8368}, year = {1997}, date = {1997-02-01}, journal = {Protein science}, volume = {6}, pages = {347-54}, abstract = {To gain insight into the free energy changes accompanying protein hydrophobic core formation, we have used computer simulations to study the formation of small clusters of nonpolar solutes in water. A barrier to association is observed at the largest solute separation that does not allow substantial solvent penetration. The barrier reflects an effective increase in the size of the cavity occupied by the expanded but water-excluding cluster relative to both the close-packed cluster and the fully solvated separated solutes; a similar effect may contribute to the barrier to protein folding/unfolding. Importantly for the simulation of protein folding without explicit solvent, we find that the interactions between nonpolar solutes of varying size and number can be approximated by a linear function of the molecular surface, but not the solvent-accessible surface of the solutes. Comparison of the free energy of cluster formation to that of dimer formation suggests that the assumption of pair additivity implicit in current protein database derived potentials may be in error.}, keywords = {}, pubstate = {published}, tppubtype = {article} } To gain insight into the free energy changes accompanying protein hydrophobic core formation, we have used computer simulations to study the formation of small clusters of nonpolar solutes in water. A barrier to association is observed at the largest solute separation that does not allow substantial solvent penetration. The barrier reflects an effective increase in the size of the cavity occupied by the expanded but water-excluding cluster relative to both the close-packed cluster and the fully solvated separated solutes; a similar effect may contribute to the barrier to protein folding/unfolding. Importantly for the simulation of protein folding without explicit solvent, we find that the interactions between nonpolar solutes of varying size and number can be approximated by a linear function of the molecular surface, but not the solvent-accessible surface of the solutes. Comparison of the free energy of cluster formation to that of dimer formation suggests that the assumption of pair additivity implicit in current protein database derived potentials may be in error. |
Bystroff, C; Baker, David Blind predictions of local protein structure in CASP2 targets using the I-sites library Journal Article Proteins, Suppl 1 , pp. 167-71, 1997, ISSN: 0887-3585. @article{30, title = {Blind predictions of local protein structure in CASP2 targets using the I-sites library}, author = { C Bystroff and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/bystroff97A.pdf}, issn = {0887-3585}, year = {1997}, date = {1997-00-01}, journal = {Proteins}, volume = {Suppl 1}, pages = {167-71}, abstract = {Blind predictions of the local structure of nine CASP2 targets were made using the I-sites library of short sequence--structure motifs, revealing strengths and weaknesses in this new knowledge-based method. Many turns between secondary structural elements were accurately predicted. Estimates of the confidence of prediction correlated well with the accuracy over the whole set. Bias toward structures used to develop the library was minimal, probably because of the extensive use of cross-validation. However, helix positions were better predicted by the PHD program. The method is likely to be sensitive to the quality of the sequence alignment. A general measure for evaluating local structure predictions is suggested.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Blind predictions of the local structure of nine CASP2 targets were made using the I-sites library of short sequence--structure motifs, revealing strengths and weaknesses in this new knowledge-based method. Many turns between secondary structural elements were accurately predicted. Estimates of the confidence of prediction correlated well with the accuracy over the whole set. Bias toward structures used to develop the library was minimal, probably because of the extensive use of cross-validation. However, helix positions were better predicted by the PHD program. The method is likely to be sensitive to the quality of the sequence alignment. A general measure for evaluating local structure predictions is suggested. |
Yi, Q; Scalley, M L; Simons, K T; Gladwin, S T; Baker, David Characterization of the free energy spectrum of peptostreptococcal protein L Journal Article Folding & design, 2 , pp. 271-80, 1997, ISSN: 1359-0278. @article{27, title = {Characterization of the free energy spectrum of peptostreptococcal protein L}, author = { Q Yi and M L Scalley and K T Simons and S T Gladwin and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/yi97A.pdf}, issn = {1359-0278}, year = {1997}, date = {1997-00-01}, journal = {Folding & design}, volume = {2}, pages = {271-80}, abstract = {BACKGROUND: Native state hydrogen/deuterium exchange studies on cytochrome c and RNase H revealed the presence of excited states with partially formed native structure. We set out to determine whether such excited states are populated for a very small and simple protein, the IgG-binding domain of peptostreptococcal protein L. RESULTS: Hydrogen/deuterium exchange data on protein L in 0-1.2 M guanidine fit well to a simple model in which the only contributions to exchange are denaturant-independent local fluctuations and global unfolding. A substantial discrepancy emerged between unfolding free energy estimates from hydrogen/deuterium exchange and linear extrapolation of earlier guanidine denaturation experiments. A better determined estimate of the free energy of unfolding obtained by global analysis of a series of thermal denaturation experiments in the presence of 0-3 M guanidine was in good agreement with the estimate from hydrogen/deuterium exchange. CONCLUSIONS: For protein L under native conditions, there do not appear to be partially folded states with free energies intermediate between that of the folded and unfolded states. The linear extrapolation method significantly underestimates the free energy of folding of protein L due to deviations from linearity in the dependence of the free energy on the denaturant concentration.}, keywords = {}, pubstate = {published}, tppubtype = {article} } BACKGROUND: Native state hydrogen/deuterium exchange studies on cytochrome c and RNase H revealed the presence of excited states with partially formed native structure. We set out to determine whether such excited states are populated for a very small and simple protein, the IgG-binding domain of peptostreptococcal protein L. RESULTS: Hydrogen/deuterium exchange data on protein L in 0-1.2 M guanidine fit well to a simple model in which the only contributions to exchange are denaturant-independent local fluctuations and global unfolding. A substantial discrepancy emerged between unfolding free energy estimates from hydrogen/deuterium exchange and linear extrapolation of earlier guanidine denaturation experiments. A better determined estimate of the free energy of unfolding obtained by global analysis of a series of thermal denaturation experiments in the presence of 0-3 M guanidine was in good agreement with the estimate from hydrogen/deuterium exchange. CONCLUSIONS: For protein L under native conditions, there do not appear to be partially folded states with free energies intermediate between that of the folded and unfolded states. The linear extrapolation method significantly underestimates the free energy of folding of protein L due to deviations from linearity in the dependence of the free energy on the denaturant concentration. |
1996 |
Bystroff, C; Simons, K T; Han, K F; Baker, David Local sequence-structure correlations in proteins Journal Article Current opinion in biotechnology, 7 , pp. 417-21, 1996, ISSN: 0958-1669. @article{214, title = {Local sequence-structure correlations in proteins}, author = { C Bystroff and K T Simons and K F Han and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/bystroff96A.pdf}, issn = {0958-1669}, year = {1996}, date = {1996-08-01}, journal = {Current opinion in biotechnology}, volume = {7}, pages = {417-21}, abstract = {Considerable progress has been made in understanding the relationship between local amino acid sequence and local protein structure. Recent highlights include numerous studies of the structures adopted by short peptides, new approaches to correlating sequence patterns with structure patterns, and folding simulations using simple potentials.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Considerable progress has been made in understanding the relationship between local amino acid sequence and local protein structure. Recent highlights include numerous studies of the structures adopted by short peptides, new approaches to correlating sequence patterns with structure patterns, and folding simulations using simple potentials. |
Han, K F; Baker, David Global properties of the mapping between local amino acid sequence and local structure in proteins Journal Article Proceedings of the National Academy of Sciences of the United States of America, 93 , pp. 5814-8, 1996, ISSN: 0027-8424. @article{215, title = {Global properties of the mapping between local amino acid sequence and local structure in proteins}, author = { K F Han and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/han96A.pdf}, issn = {0027-8424}, year = {1996}, date = {1996-06-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {93}, pages = {5814-8}, abstract = {Local protein structure prediction efforts have consistently failed to exceed approximately 70% accuracy. We characterize the degeneracy of the mapping from local sequence to local structure responsible for this failure by investigating the extent to which similar sequence segments found in different proteins adopt similar three-dimensional structures. Sequence segments 3-15 residues in length from 154 different protein families are partitioned into neighborhoods containing segments with similar sequences using cluster analysis. The consistency of the sequence-to-structure mapping is assessed by comparing the local structures adopted by sequence segments in the same neighborhood in proteins of known structure. In the 154 families, 45% and 28% of the positions occur in neighborhoods in which one and two local structures predominate, respectively. The sequence patterns that characterize the neighborhoods in the first class probably include virtually all of the short sequence motifs in proteins that consistently occur in a particular local structure. These patterns, many of which occur in transitions between secondary structural elements, are an interesting combination of previously studied and novel motifs. The identification of sequence patterns that consistently occur in one or a small number of local structures in proteins should contribute to the prediction of protein structure from sequence.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Local protein structure prediction efforts have consistently failed to exceed approximately 70% accuracy. We characterize the degeneracy of the mapping from local sequence to local structure responsible for this failure by investigating the extent to which similar sequence segments found in different proteins adopt similar three-dimensional structures. Sequence segments 3-15 residues in length from 154 different protein families are partitioned into neighborhoods containing segments with similar sequences using cluster analysis. The consistency of the sequence-to-structure mapping is assessed by comparing the local structures adopted by sequence segments in the same neighborhood in proteins of known structure. In the 154 families, 45% and 28% of the positions occur in neighborhoods in which one and two local structures predominate, respectively. The sequence patterns that characterize the neighborhoods in the first class probably include virtually all of the short sequence motifs in proteins that consistently occur in a particular local structure. These patterns, many of which occur in transitions between secondary structural elements, are an interesting combination of previously studied and novel motifs. The identification of sequence patterns that consistently occur in one or a small number of local structures in proteins should contribute to the prediction of protein structure from sequence. |
Yi, Q; Baker, David Direct evidence for a two-state protein unfolding transition from hydrogen-deuterium exchange, mass spectrometry, and NMR Journal Article Protein science, 5 , pp. 1060-6, 1996, ISSN: 0961-8368. @article{24, title = {Direct evidence for a two-state protein unfolding transition from hydrogen-deuterium exchange, mass spectrometry, and NMR}, author = { Q Yi and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/yi96A.pdf}, issn = {0961-8368}, year = {1996}, date = {1996-06-01}, journal = {Protein science}, volume = {5}, pages = {1060-6}, abstract = {We use mass spectrometry in conjunction with hydrogen-deuterium exchange and NMR to characterize the conformational dynamics of the 62-residue IgG binding domain of protein L under conditions in which the native state is marginally stable. Mass spectra of protein L after short incubations in D2O reveal the presence of two distinct populations containing different numbers of protected protons. NMR experiments indicate that protons in the hydrophobic core are protected in one population, whereas all protons are exchanged for deuterons in the other. As the exchange period is increased, molecules are transferred from the former population to the latter. The absence of molecules with a subset of the core protons protected suggests that exchange occurs in part via a highly concerted transition to an excited state in which all protons exchange rapidly with deuterons. A steady increase in the molecular weight of the population with protected protons, and variation in the exchange rates of the individual protected protons indicates the presence of an additional exchange mechanism. A simple model in which exchange results from rapid (> 10(5)/s) local fluctuations around the native state superimposed upon transitions to an unfolded excited state at approximately 0.06/s is supported by qualitative agreement between the observed mass spectra and the mass spectra simulated according to the model using NMR-derived estimates of the proton exchange rates.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We use mass spectrometry in conjunction with hydrogen-deuterium exchange and NMR to characterize the conformational dynamics of the 62-residue IgG binding domain of protein L under conditions in which the native state is marginally stable. Mass spectra of protein L after short incubations in D2O reveal the presence of two distinct populations containing different numbers of protected protons. NMR experiments indicate that protons in the hydrophobic core are protected in one population, whereas all protons are exchanged for deuterons in the other. As the exchange period is increased, molecules are transferred from the former population to the latter. The absence of molecules with a subset of the core protons protected suggests that exchange occurs in part via a highly concerted transition to an excited state in which all protons exchange rapidly with deuterons. A steady increase in the molecular weight of the population with protected protons, and variation in the exchange rates of the individual protected protons indicates the presence of an additional exchange mechanism. A simple model in which exchange results from rapid (> 10(5)/s) local fluctuations around the native state superimposed upon transitions to an unfolded excited state at approximately 0.06/s is supported by qualitative agreement between the observed mass spectra and the mass spectra simulated according to the model using NMR-derived estimates of the proton exchange rates. |
1995 |
Han, K F; Baker, David Recurring local sequence motifs in proteins Journal Article Journal of molecular biology, 251 , pp. 176-87, 1995, ISSN: 0022-2836. @article{22, title = {Recurring local sequence motifs in proteins}, author = { K F Han and David Baker}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/han95A.pdf}, issn = {0022-2836}, year = {1995}, date = {1995-08-01}, journal = {Journal of molecular biology}, volume = {251}, pages = {176-87}, abstract = {We describe a completely automated approach to identifying local sequence motifs that transcend protein family boundaries. Cluster analysis is used to identify recurring patterns of variation at single positions and in short segments of contiguous positions in multiple sequence alignments for a non-redundant set of protein families. Parallel experiments on simulated data sets constructed with the overall residue frequencies of proteins but not the inter-residue correlations show that naturally occurring protein sequences are significantly more clustered than the corresponding random sequences for window lengths ranging from one to 13 contiguous positions. The patterns of variation at single positions are not in general surprising: chemically similar amino acids tend to be grouped together. More interesting patterns emerge as the window length increases. The patterns of variation for longer window lengths are in part recognizable patterns of hydrophobic and hydrophilic residues, and in part less obvious combinations. A particularly interesting class of patterns features highly conserved glycine residues. The patterns provide a means to abstract the information contained in multiple sequence alignments and may be useful for comparison of distantly related sequences or sequence families and for protein structure prediction.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe a completely automated approach to identifying local sequence motifs that transcend protein family boundaries. Cluster analysis is used to identify recurring patterns of variation at single positions and in short segments of contiguous positions in multiple sequence alignments for a non-redundant set of protein families. Parallel experiments on simulated data sets constructed with the overall residue frequencies of proteins but not the inter-residue correlations show that naturally occurring protein sequences are significantly more clustered than the corresponding random sequences for window lengths ranging from one to 13 contiguous positions. The patterns of variation at single positions are not in general surprising: chemically similar amino acids tend to be grouped together. More interesting patterns emerge as the window length increases. The patterns of variation for longer window lengths are in part recognizable patterns of hydrophobic and hydrophilic residues, and in part less obvious combinations. A particularly interesting class of patterns features highly conserved glycine residues. The patterns provide a means to abstract the information contained in multiple sequence alignments and may be useful for comparison of distantly related sequences or sequence families and for protein structure prediction. |
Gu, H; Yi, Q; Bray, S T; Riddle, D S; Shiau, A K; Baker, David A phage display system for studying the sequence determinants of protein folding Journal Article Protein science, 4 , pp. 1108-17, 1995, ISSN: 0961-8368. @article{216, title = {A phage display system for studying the sequence determinants of protein folding}, author = { H Gu and Q Yi and S T Bray and D S Riddle and A K Shiau and David Baker}, issn = {0961-8368}, year = {1995}, date = {1995-06-01}, journal = {Protein science}, volume = {4}, pages = {1108-17}, abstract = {We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein. |
1994 |
Baker, D; Agard, D A Influenza hemagglutinin: kinetic control of protein function. Journal Article Structure (London, England : 1993), 2 , pp. 907-10, 1994, ISSN: 0969-2126. @article{572, title = {Influenza hemagglutinin: kinetic control of protein function.}, author = { D Baker and D A Agard}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/influenzahemagglutinin_Baker1994.pdf}, issn = {0969-2126}, year = {1994}, date = {1994-10-01}, journal = {Structure (London, England : 1993)}, volume = {2}, pages = {907-10}, abstract = {In response to decreased pH, influenza hemagglutinin changes to a more stable conformation. Such changes, which can be controlled thermodynamically or kinetically, are the method by which many biological textquoterightswitchestextquoteright are thrown.}, keywords = {}, pubstate = {published}, tppubtype = {article} } In response to decreased pH, influenza hemagglutinin changes to a more stable conformation. Such changes, which can be controlled thermodynamically or kinetically, are the method by which many biological textquoterightswitchestextquoteright are thrown. |
Baker, D; Agard, D A Kinetics versus thermodynamics in protein folding. Journal Article Biochemistry, 33 , pp. 7505-9, 1994, ISSN: 0006-2960. @article{571, title = {Kinetics versus thermodynamics in protein folding.}, author = { D Baker and D A Agard}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/kineticsversusthermodynamics_Baker1994.pdf}, issn = {0006-2960}, year = {1994}, date = {1994-06-01}, journal = {Biochemistry}, volume = {33}, pages = {7505-9}, abstract = {Until quite recently it has been generally believed that the observed tertiary structure of a protein is controlled by thermodynamic and not kinetic processes. In this essay we review several recent results which call into question the universality of the thermodynamic hypothesis and discuss their implications for the understanding of protein folding.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Until quite recently it has been generally believed that the observed tertiary structure of a protein is controlled by thermodynamic and not kinetic processes. In this essay we review several recent results which call into question the universality of the thermodynamic hypothesis and discuss their implications for the understanding of protein folding. |
1993 |
Baker, D; Shiau, A K; Agard, D A The role of pro regions in protein folding. Journal Article Current Opinion in Cell Biology, 5 , pp. 966-70, 1993, ISSN: 0955-0674. @article{570, title = {The role of pro regions in protein folding.}, author = { D Baker and A K Shiau and D A Agard}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/theroleofproregions_Baker1993.pdf}, issn = {0955-0674}, year = {1993}, date = {1993-12-01}, journal = {Current Opinion in Cell Biology}, volume = {5}, pages = {966-70}, abstract = {In vivo, many proteases are synthesized as larger precursors. During the maturation process, the catalytically active protease domain is released from the larger polypeptide or pro-enzyme by one or more proteolytic processing steps. In several well studied cases, amino-terminal pro regions have been shown to play a fundamental role in the folding of the associated protease domains. The mechanism by which pro regions facilitate folding appears to be significantly different from that used by the molecular chaperones. Recent results suggest that the pro region assisted folding mechanism may be used by a wide variety of proteases, and perhaps even by non-proteases.}, keywords = {}, pubstate = {published}, tppubtype = {article} } In vivo, many proteases are synthesized as larger precursors. During the maturation process, the catalytically active protease domain is released from the larger polypeptide or pro-enzyme by one or more proteolytic processing steps. In several well studied cases, amino-terminal pro regions have been shown to play a fundamental role in the folding of the associated protease domains. The mechanism by which pro regions facilitate folding appears to be significantly different from that used by the molecular chaperones. Recent results suggest that the pro region assisted folding mechanism may be used by a wide variety of proteases, and perhaps even by non-proteases. |
Bystroff, C; Baker, D; Fletterick, R J; Agard, D A PRISM: application to the solution of two protein structures Journal Article Acta crystallographica. Section D, 49 , pp. 440-8, 1993, ISSN: 0907-4449. @article{326, title = {PRISM: application to the solution of two protein structures}, author = { C Bystroff and D Baker and R J Fletterick and D A Agard}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/bystroff93A.pdf}, issn = {0907-4449}, year = {1993}, date = {1993-09-01}, journal = {Acta crystallographica. Section D}, volume = {49}, pages = {440-8}, abstract = {The previous paper described a phase-refinement strategy for protein crystallography which exploited the information that proteins consist of connected linear chains of atoms. Here the method is applied to a molecular-replacement problem, the structure of the protease inhibitor ecotin bound to trypsin, and a single isomorphous replacement problem, the structure of the N-terminal domain of apolipoprotein E. The starting phases for the ecotin-trypsin complex were based on a partial model (trypsin) containing 61% of the atoms in the complex. Iterative skeletonization gave better results than either solvent flattening or twofold non-crystallographic symmetry averaging as measured by the reduction in the free R factor [Br"unger (1992). Nature (London), 355, 472-474]. Protection of the trypsin density during the course of the refinement greatly improved the performance of both skeletonizing and solvent flattening. In the case of apolipoprotein E, previous attempts using solvent flattening had failed to improve the SIR phases to the point of obtaining an interpretable map. The combination of iterative skeletonization and solvent flattening decreased the phase error with respect to the final refined structure, significantly more than solvent flattening alone. The final maps generated by the skeletonization procedure for both the ecotin-trypsin complex and apolipoprotein E were readily interpretable.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The previous paper described a phase-refinement strategy for protein crystallography which exploited the information that proteins consist of connected linear chains of atoms. Here the method is applied to a molecular-replacement problem, the structure of the protease inhibitor ecotin bound to trypsin, and a single isomorphous replacement problem, the structure of the N-terminal domain of apolipoprotein E. The starting phases for the ecotin-trypsin complex were based on a partial model (trypsin) containing 61% of the atoms in the complex. Iterative skeletonization gave better results than either solvent flattening or twofold non-crystallographic symmetry averaging as measured by the reduction in the free R factor [Br"unger (1992). Nature (London), 355, 472-474]. Protection of the trypsin density during the course of the refinement greatly improved the performance of both skeletonizing and solvent flattening. In the case of apolipoprotein E, previous attempts using solvent flattening had failed to improve the SIR phases to the point of obtaining an interpretable map. The combination of iterative skeletonization and solvent flattening decreased the phase error with respect to the final refined structure, significantly more than solvent flattening alone. The final maps generated by the skeletonization procedure for both the ecotin-trypsin complex and apolipoprotein E were readily interpretable. |
Baker, D; Bystroff, C; Fletterick, R J; Agard, D A PRISM: topologically constrained phased refinement for macromolecular crystallography Journal Article Acta crystallographica. Section D, 49 , pp. 429-39, 1993, ISSN: 0907-4449. @article{323, title = {PRISM: topologically constrained phased refinement for macromolecular crystallography}, author = { D Baker and C Bystroff and R J Fletterick and D A Agard}, issn = {0907-4449}, year = {1993}, date = {1993-09-01}, journal = {Acta crystallographica. Section D}, volume = {49}, pages = {429-39}, abstract = {We describe the further development of phase refinement by iterative skeletonization (PRISM), a recently introduced phase-refinement strategy [Wilson & Agard (1993). Acta Cryst. A49, 97-104] which makes use of the information that proteins consist of connected linear chains of atoms. An initial electron-density map is generated with inaccurate phases derived from a partial structure or from isomorphous replacement. A linear connected skeleton is then constructed from the map using a modified version of Greertextquoterights algorithm [Greer (1985). Methods Enzymol. 115, 206-226] and a new map is created from the skeleton. This textquoterightskeletonizedtextquoteright map is Fourier transformed to obtained new phases, which are combined with any starting-phase information and the experimental structure-factor amplitudes to produce a new map. The procedure is iterated until convergence is reached. In this paper significant improvements to the method are described as is a challenging molecular-replacement test case in which initial phases are calculated from a model containing only one third of the atoms of the intact protein. Application of the skeletonization procedure yields an easily interpretable map. In contrast, application of solvent flattening does not significantly improve the starting map. The iterative skeletonization procedure performs well in the presence of random noise and missing data, but requires Fourier data to at least 3.0 A. The constraints of linearity and connectedness prove strong enough to restore not only missing phase information, but also missing amplitudes. This enables the use of a powerful statistical test, analogous to the textquoterightfree R factortextquoteright of conventional refinement [Br"unger (1992). Nature (London), 355, 472-474], for optimizing the performance of the skeletonization procedure. In the accompanying paper, we describe the application of the method to the solution of the structure of the protease inhibitor ecotin bound to trypsin and to a single isomorphous replacement problem.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe the further development of phase refinement by iterative skeletonization (PRISM), a recently introduced phase-refinement strategy [Wilson & Agard (1993). Acta Cryst. A49, 97-104] which makes use of the information that proteins consist of connected linear chains of atoms. An initial electron-density map is generated with inaccurate phases derived from a partial structure or from isomorphous replacement. A linear connected skeleton is then constructed from the map using a modified version of Greertextquoterights algorithm [Greer (1985). Methods Enzymol. 115, 206-226] and a new map is created from the skeleton. This textquoterightskeletonizedtextquoteright map is Fourier transformed to obtained new phases, which are combined with any starting-phase information and the experimental structure-factor amplitudes to produce a new map. The procedure is iterated until convergence is reached. In this paper significant improvements to the method are described as is a challenging molecular-replacement test case in which initial phases are calculated from a model containing only one third of the atoms of the intact protein. Application of the skeletonization procedure yields an easily interpretable map. In contrast, application of solvent flattening does not significantly improve the starting map. The iterative skeletonization procedure performs well in the presence of random noise and missing data, but requires Fourier data to at least 3.0 A. The constraints of linearity and connectedness prove strong enough to restore not only missing phase information, but also missing amplitudes. This enables the use of a powerful statistical test, analogous to the textquoterightfree R factortextquoteright of conventional refinement [Br"unger (1992). Nature (London), 355, 472-474], for optimizing the performance of the skeletonization procedure. In the accompanying paper, we describe the application of the method to the solution of the structure of the protease inhibitor ecotin bound to trypsin and to a single isomorphous replacement problem. |
Ruohola-Baker, H; Grell, E; Chou, T B; Baker, D; Jan, L Y; Jan, Y N Spatially localized rhomboid is required for establishment of the dorsal-ventral axis in Drosophila oogenesis Journal Article Cell, 73 , pp. 953-65, 1993, ISSN: 0092-8674. @article{327, title = {Spatially localized rhomboid is required for establishment of the dorsal-ventral axis in Drosophila oogenesis}, author = { H Ruohola-Baker and E Grell and T B Chou and D Baker and L Y Jan and Y N Jan}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/ruohola-baker93A-1.pdf}, issn = {0092-8674}, year = {1993}, date = {1993-06-01}, journal = {Cell}, volume = {73}, pages = {953-65}, abstract = {The establishment of dorsal-ventral asymmetry of the Drosophila embryo requires a group of genes that act maternally. None of the previously identified dorsal-ventral axis genes are known to produce asymmetrically localized gene products during oogenesis. We show that rhomboid (rho), a novel member of this group, encodes a protein that is localized on the apical surface of the dorsal-anterior follicle cells surrounding the oocyte. Loss of rho function causes ventralization of the eggshell and the embryo, whereas ectopic expression leads to dorsalization of both structures. Thus, spatially restricted rho is necessary and sufficient for dorsal-ventral axis formation. We propose, based on these observations and double mutant experiments, that the spatially restricted rho protein leads to selective activation of the epidermal growth factor receptor in the dorsal follicle cells and subsequently the specification of the dorsal follicle cells.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The establishment of dorsal-ventral asymmetry of the Drosophila embryo requires a group of genes that act maternally. None of the previously identified dorsal-ventral axis genes are known to produce asymmetrically localized gene products during oogenesis. We show that rhomboid (rho), a novel member of this group, encodes a protein that is localized on the apical surface of the dorsal-anterior follicle cells surrounding the oocyte. Loss of rho function causes ventralization of the eggshell and the embryo, whereas ectopic expression leads to dorsalization of both structures. Thus, spatially restricted rho is necessary and sufficient for dorsal-ventral axis formation. We propose, based on these observations and double mutant experiments, that the spatially restricted rho protein leads to selective activation of the epidermal growth factor receptor in the dorsal follicle cells and subsequently the specification of the dorsal follicle cells. |
Baker, D; Krukowski, A E; Agard, D A Uniqueness and the ab initio phase problem in macromolecular crystallography Journal Article Acta crystallographica. Section D, 49 , pp. 186-92, 1993, ISSN: 0907-4449. @article{325, title = {Uniqueness and the ab initio phase problem in macromolecular crystallography}, author = { D Baker and A E Krukowski and D A Agard}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/baker93C.pdf}, issn = {0907-4449}, year = {1993}, date = {1993-01-01}, journal = {Acta crystallographica. Section D}, volume = {49}, pages = {186-92}, abstract = {The crystallographic phase problem is indeterminate in the absence of additional chemical information. A successful ab initio approach to the macromolecular phase problem must employ sufficient chemical constraints to limit the solutions to a manageably small number. Here we show that commonly employed chemical constraints - positivity, atomicity and a solvent boundary - leave the phase problem greatly underdetermined for Fourier data sets of moderate (2.5-3.0 A) resolution. Entropy maximization is also beset by multiple false solutions: electron-density maps are readily generated which satisfy the same Fourier amplitude constraints but have higher entropies than the true solution. We conclude that a successful ab initio approach must make use of high-resolution Fourier data and/or stronger chemical constraints. One such constraint is the connectivity of the macromolecule. We describe a rapid algorithm for measuring the connectivity of a map, and show its utility in reducing the multiplicity of solutions to the phase problem.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The crystallographic phase problem is indeterminate in the absence of additional chemical information. A successful ab initio approach to the macromolecular phase problem must employ sufficient chemical constraints to limit the solutions to a manageably small number. Here we show that commonly employed chemical constraints - positivity, atomicity and a solvent boundary - leave the phase problem greatly underdetermined for Fourier data sets of moderate (2.5-3.0 A) resolution. Entropy maximization is also beset by multiple false solutions: electron-density maps are readily generated which satisfy the same Fourier amplitude constraints but have higher entropies than the true solution. We conclude that a successful ab initio approach must make use of high-resolution Fourier data and/or stronger chemical constraints. One such constraint is the connectivity of the macromolecule. We describe a rapid algorithm for measuring the connectivity of a map, and show its utility in reducing the multiplicity of solutions to the phase problem. |
D, Baker; HS, Chan; KA, Dill Coordinate-Space Formulation of Polymer Lattice Cluster Theory Journal Article Journal of Chemical Physics, 1993. @article{324, title = {Coordinate-Space Formulation of Polymer Lattice Cluster Theory}, author = { Baker D and Chan HS and Dill KA}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/baker93B.pdf}, year = {1993}, date = {1993-01-01}, journal = {Journal of Chemical Physics}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
1992 |
Baker, D; Silen, J L; Agard, D A Protease pro region required for folding is a potent inhibitor of the mature enzyme Journal Article Proteins, 12 , pp. 339-44, 1992, ISSN: 0887-3585. @article{328, title = {Protease pro region required for folding is a potent inhibitor of the mature enzyme}, author = { D Baker and J L Silen and D A Agard}, issn = {0887-3585}, year = {1992}, date = {1992-04-01}, journal = {Proteins}, volume = {12}, pages = {339-44}, abstract = {alpha-Lytic protease, an extracellular bacterial serine protease, is synthesized with a large pro region that is required in vivo for the proper folding of the protease domain. To allow detailed mechanistic study, we have reconstituted pro region-dependent folding in vitro. The pro region promotes folding of the protease domain in the absence of other protein factors or exogenous energy sources. Surprisingly, we find that the pro region is a high affinity inhibitor of the mature protease. The pro region also inhibits the closely related Streptomyces griseus protease B, but not the more distantly related, yet structurally similar protease, elastase. Based on these data, we suggest a mechanism in which pro region binding reduces the free energy of a late folding transition state having native-like structure.}, keywords = {}, pubstate = {published}, tppubtype = {article} } alpha-Lytic protease, an extracellular bacterial serine protease, is synthesized with a large pro region that is required in vivo for the proper folding of the protease domain. To allow detailed mechanistic study, we have reconstituted pro region-dependent folding in vitro. The pro region promotes folding of the protease domain in the absence of other protein factors or exogenous energy sources. Surprisingly, we find that the pro region is a high affinity inhibitor of the mature protease. The pro region also inhibits the closely related Streptomyces griseus protease B, but not the more distantly related, yet structurally similar protease, elastase. Based on these data, we suggest a mechanism in which pro region binding reduces the free energy of a late folding transition state having native-like structure. |
Baker, D; Sohl, J L; Agard, D A A protein-folding reaction under kinetic control Journal Article Nature, 356 , pp. 263-5, 1992, ISSN: 0028-0836. @article{329, title = {A protein-folding reaction under kinetic control}, author = { D Baker and J L Sohl and D A Agard}, issn = {0028-0836}, year = {1992}, date = {1992-03-01}, journal = {Nature}, volume = {356}, pages = {263-5}, abstract = {Synthesis of alpha-lytic protease is as a precursor containing a 166 amino-acid pro region transiently required for the correct folding of the protease domain. By omitting the pro region in an in vitro refolding reaction we trapped an inactive, but folding competent state (I) having an expanded radius yet native-like secondary structure. The I state is stable for weeks at physiological pH in the absence of denaturant, but rapidly folds to the active, native state on addition of the pro region as a separate polypeptide chain. The mechanism of action of the pro region is distinct from that of the chaperonins: rather than reducing the rate of off-pathway reactions, the pro region accelerates the rate-limiting step on the folding pathway by more than 10(7). Because both the I and native states are stable under identical conditions with no detectable interconversion, the folding of alpha-lytic protease must be under kinetic and not thermodynamic control.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Synthesis of alpha-lytic protease is as a precursor containing a 166 amino-acid pro region transiently required for the correct folding of the protease domain. By omitting the pro region in an in vitro refolding reaction we trapped an inactive, but folding competent state (I) having an expanded radius yet native-like secondary structure. The I state is stable for weeks at physiological pH in the absence of denaturant, but rapidly folds to the active, native state on addition of the pro region as a separate polypeptide chain. The mechanism of action of the pro region is distinct from that of the chaperonins: rather than reducing the rate of off-pathway reactions, the pro region accelerates the rate-limiting step on the folding pathway by more than 10(7). Because both the I and native states are stable under identical conditions with no detectable interconversion, the folding of alpha-lytic protease must be under kinetic and not thermodynamic control. |
1991 |
Ruohola, H; Bremer, K A; Baker, D; Swedlow, J R; Jan, L Y; Jan, Y N Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila. Journal Article Cell, 66 , pp. 433-49, 1991, ISSN: 0092-8674. @article{330, title = {Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila.}, author = { H Ruohola and K A Bremer and D Baker and J R Swedlow and L Y Jan and Y N Jan}, issn = {0092-8674}, year = {1991}, date = {1991-08-01}, journal = {Cell}, volume = {66}, pages = {433-49}, abstract = {Oogenesis in Drosophila involves specification of both germ cells and the surrounding somatic follicle cells, as well as the determination of oocyte polarity. We found that two neurogenic genes, Notch and Delta, are required in oogenesis. These genes encode membrane proteins with epidermal growth factor repeats and are essential in the decision of an embryonic ectodermal cell to take on the fate of neuroblast or epidermoblast. In oogenesis, mutation in either gene leads to an excess of posterior follicle cells, a cell fate change reminiscent of the hyperplasia of neuroblasts seen in neurogenic mutant embryos. Furthermore, the Notch mutation in somatic cells causes mislocalization of bicoid in the oocyte. These results suggest that the neurogenic genes Notch and Delta are involved in both follicle cell development and the establishment of anterior-posterior polarity in the oocyte.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Oogenesis in Drosophila involves specification of both germ cells and the surrounding somatic follicle cells, as well as the determination of oocyte polarity. We found that two neurogenic genes, Notch and Delta, are required in oogenesis. These genes encode membrane proteins with epidermal growth factor repeats and are essential in the decision of an embryonic ectodermal cell to take on the fate of neuroblast or epidermoblast. In oogenesis, mutation in either gene leads to an excess of posterior follicle cells, a cell fate change reminiscent of the hyperplasia of neuroblasts seen in neurogenic mutant embryos. Furthermore, the Notch mutation in somatic cells causes mislocalization of bicoid in the oocyte. These results suggest that the neurogenic genes Notch and Delta are involved in both follicle cell development and the establishment of anterior-posterior polarity in the oocyte. |
1990 |
Baker, D; Wuestehube, L; Schekman, R; Botstein, D; Segev, N GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction Journal Article Proceedings of the National Academy of Sciences of the United States of America, 87 , pp. 355-9, 1990, ISSN: 0027-8424. @article{331, title = {GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction}, author = { D Baker and L Wuestehube and R Schekman and D Botstein and N Segev}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/baker90A.pdf}, issn = {0027-8424}, year = {1990}, date = {1990-01-01}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {87}, pages = {355-9}, abstract = {The 21-kDa GTP-binding Ypt1 protein (Ypt1p) is required for protein transport from the endoplasmic reticulum to the Golgi complex in yeast extracts. Ypt1 antibodies block transport; this inhibition is alleviated by competition with excess purified Ypt1p produced in bacteria. Furthermore, extracts of cells carrying the mutation ypt1-1 are defective in transport, but transport is restored if a cytosolic fraction from wild-type cells is provided. The in vitro transport reaction also requires physiological levels of Ca2+. However, Ypt1p functions independently of Ca2+. First, buffering the free Ca2+ at concentrations ranging from 1 nM to 10 microM does not relieve inhibition by Ypt1 antibodies. Second, consumption of a Ca2+-requiring intermediate that accumulates in Ca2+-deficient incubations is not inhibited by anti-Ypt1 antibodies, although completion of transport requires ATP and an N-ethylmaleimide-sensitive factor. Thus, Ypt1p and Ca2+ are required at distinct steps.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The 21-kDa GTP-binding Ypt1 protein (Ypt1p) is required for protein transport from the endoplasmic reticulum to the Golgi complex in yeast extracts. Ypt1 antibodies block transport; this inhibition is alleviated by competition with excess purified Ypt1p produced in bacteria. Furthermore, extracts of cells carrying the mutation ypt1-1 are defective in transport, but transport is restored if a cytosolic fraction from wild-type cells is provided. The in vitro transport reaction also requires physiological levels of Ca2+. However, Ypt1p functions independently of Ca2+. First, buffering the free Ca2+ at concentrations ranging from 1 nM to 10 microM does not relieve inhibition by Ypt1 antibodies. Second, consumption of a Ca2+-requiring intermediate that accumulates in Ca2+-deficient incubations is not inhibited by anti-Ypt1 antibodies, although completion of transport requires ATP and an N-ethylmaleimide-sensitive factor. Thus, Ypt1p and Ca2+ are required at distinct steps. |
1989 |
Baker, D; Schekman, R Reconstitution of protein transport using broken yeast spheroplasts Journal Article Methods in Cell Biology, 31 , pp. 127-41, 1989, ISSN: 0091-679X. @article{569, title = {Reconstitution of protein transport using broken yeast spheroplasts}, author = { D Baker and R Schekman}, issn = {0091-679X}, year = {1989}, date = {1989-00-01}, journal = {Methods in Cell Biology}, volume = {31}, pages = {127-41}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
1988 |
Baker, D; Hicke, L; Rexach, M; Schleyer, M; Schekman, R Reconstitution of SEC gene product-dependent intercompartmental protein transport Journal Article Cell, 54 , pp. 335-44, 1988, ISSN: 0092-8674. @article{332, title = {Reconstitution of SEC gene product-dependent intercompartmental protein transport}, author = { D Baker and L Hicke and M Rexach and M Schleyer and R Schekman}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/baker88.pdf}, issn = {0092-8674}, year = {1988}, date = {1988-07-01}, journal = {Cell}, volume = {54}, pages = {335-44}, abstract = {Transport of alpha-factor precursor from the endoplasmic reticulum to the Golgi apparatus has been reconstituted in gently lysed yeast spheroplasts. Transport is measured through the coupled addition of outer-chain carbohydrate to [35S]methionine-labeled alpha-factor precursor translocated into the endoplasmic reticulum of broken spheroplasts. The reaction is absolutely dependent on ATP, stimulated 6-fold by cytosol, and occurs between physically separable sealed compartments. Transport is inhibited by the guanine nucleotide analog GTP gamma S. sec23 mutant cells have a temperature-sensitive defect in endoplasmic reticulum-to-Golgi transport in vivo. This defect has been reproduced in vitro using sec23 membranes and cytosol. Transport at 30 degrees C with sec23 membranes requires addition of cytosol containing the SEC23 (wild-type) gene product. This demonstrates that an in vitro inter-organelle transport reaction depends on a factor required for transport in vivo. Complementation of sec mutations in vitro provides a functional assay for the purification of individual intercompartmental transport factors.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Transport of alpha-factor precursor from the endoplasmic reticulum to the Golgi apparatus has been reconstituted in gently lysed yeast spheroplasts. Transport is measured through the coupled addition of outer-chain carbohydrate to [35S]methionine-labeled alpha-factor precursor translocated into the endoplasmic reticulum of broken spheroplasts. The reaction is absolutely dependent on ATP, stimulated 6-fold by cytosol, and occurs between physically separable sealed compartments. Transport is inhibited by the guanine nucleotide analog GTP gamma S. sec23 mutant cells have a temperature-sensitive defect in endoplasmic reticulum-to-Golgi transport in vivo. This defect has been reproduced in vitro using sec23 membranes and cytosol. Transport at 30 degrees C with sec23 membranes requires addition of cytosol containing the SEC23 (wild-type) gene product. This demonstrates that an in vitro inter-organelle transport reaction depends on a factor required for transport in vivo. Complementation of sec mutations in vitro provides a functional assay for the purification of individual intercompartmental transport factors. |
Payne, G S; Baker, D; van Tuinen, E; Schekman, R Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast Journal Article The Journal of cell biology, 106 , pp. 1453-61, 1988, ISSN: 0021-9525. @article{333, title = {Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast}, author = { G S Payne and D Baker and E van Tuinen and R Schekman}, url = {https://www.bakerlab.org/wp-content/uploads/2016/06/payne88A.pdf}, issn = {0021-9525}, year = {1988}, date = {1988-05-01}, journal = {The Journal of cell biology}, volume = {106}, pages = {1453-61}, abstract = {Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells. |