@article{Hosseinzadeh2017,

title = {Comprehensive computational design of ordered peptide macrocycles},

author = {Hosseinzadeh, Parisa* and Bhardwaj, Gaurav* and Mulligan, Vikram Khipple* and Shortridge, Matthew D. and Craven, Timothy W. and Pardo-Avila, F{'a}tima and Rettie, Stephen A. and Kim, David E. and Silva, Daniel-Adriano and Ibrahim, Yehia M. and Webb, Ian K. and Cort, John R. and Adkins, Joshua N. and Varani, Gabriele and Baker, David},

url = {http://science.sciencemag.org/content/358/6369/1461

https://www.bakerlab.org/wp-content/uploads/2017/12/Science_Hosseinzadeh_et_al_2017.pdf},

doi = {10.1126/science.aap7577},

issn = {0036-8075},

year  = {2017},

date = {2017-12-15},

journal = {Science},

volume = {358},

number = {6369},

pages = {1461-1466},

abstract = {Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with L-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Butterfield2017,

title = {Evolution of a designed protein assembly encapsulating its own RNA genome},

author = {Butterfield, Gabriel L.*

and Lajoie, Marc J.*

and Gustafson, Heather H.

and Sellers, Drew L.

and Nattermann, Una

and Ellis, Daniel

and Bale, Jacob B.

and Ke, Sharon

and Lenz, Garreck H.

and Yehdego, Angelica

and Ravichandran, Rashmi

and Pun, Suzie H.

and King, Neil P.

and Baker, David},

url = {http://dx.doi.org/10.1038/nature25157

https://www.bakerlab.org/wp-content/uploads/2017/12/Nature_Butterfield_et_al_2017.pdf},

doi = {10.1038/nature25157},

issn = {1476-4687},

year  = {2017},

date = {2017-12-13},

journal = {Nature},

abstract = {The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a ‘blank slate’ to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6hours of treatment), and in vivo circulation time (from less than 5minutes to approximately 4.5hours). The resulting synthetic nucleocapsids package one full length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at ‘top-down’ modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary ‘bottom-up’ approach with considerable advantages in programmability and control.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Bhardwaj2016,

title = {Accurate de novo design of hyperstable constrained peptides},

author = { Gaurav Bhardwaj* and Vikram Khipple Mulligan* and Christopher D. Bahl* and Jason M. Gilmore and Peta J. Harvey and Olivier Cheneval and Garry W. Buchko and Surya V. S. R. K. Pulavarti and Quentin Kaas and Alexander Eletsky and Po-Ssu Huang and William A. Johnsen and Per Jr Greisen and Gabriel J. Rocklin and Yifan Song and Thomas W. Linsky and Andrew Watkins and Stephen A. Rettie and Xianzhong Xu,	Lauren P. Carter and Richard Bonneau and James M. Olson and Evangelos Coutsias and Colin E. Correnti and Thomas Szyperski and David J. Craik and David Baker	},

url = {https://www.bakerlab.org/wp-content/uploads/2016/09/Bhardwaj_Nature_2016.pdf},

doi = {10.1038/nature19791},

year  = {2016},

date = {2016-09-14},

journal = {Nature},

abstract = {Naturally occurring, pharmacologically active peptides constrained with covalent crosslinks generally have shapes that have evolved to fit precisely into binding pockets on their targets. Such peptides can have excellent pharmaceutical properties, combining the stability and tissue penetration of small-molecule drugs with the specificity of much larger protein therapeutics. The ability to design constrained peptides with precisely specified tertiary structures would enable the design of shape-complementary inhibitors of arbitrary targets. Here we describe the development of computational methods for accurate de novo design of conformationally restricted peptides, and the use of these methods to design 18–47 residue, disulfide-crosslinked peptides, a subset of which are heterochiral and/or N–C backbone-cyclized. Both genetically encodable and non-canonical peptides are exceptionally stable to thermal and chemical denaturation, and 12 experimentally determined X-ray and NMR structures are nearly identical to the computational design models. The computational design methods and stable scaffolds presented here provide the basis for development of a new generation of peptide-based drugs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{L2015,

title = {Rational design of α-helical tandem repeat proteins with closed architectures},

author = {L Doyle and J Hallinan and J Bolduc and F Parmeggiani and D Baker and BL Stoddard and P Bradley},

url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Doyle_Nature_2015.pdf},

doi = {10.1038/nature16191},

year  = {2015},

date = {2015-12-24},

journal = {Nature},

volume = {528(7583)},

pages = {585-8},

abstract = {Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks. The overall architecture of tandem repeat protein structures--which is dictated by the internal geometry and local packing of the repeat building blocks--is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed α-solenoid repeat structures (α-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the amino (N) and carboxy (C) termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed α-solenoid repeats with a left-handed helical architecture that--to our knowledge--is not yet present in the protein structure database.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{TJ2015,

title = {Exploring the repeat protein universe through computational protein design},

author = {TJ Brunette and F Parmeggiani and PS Huang and G Bhabha and DC Ekiert and SE Tsutakawa and GL Hura and JA Tainer and D Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Brunette_Nature_2015.pdf},

doi = {10.1038/nature16162},

year  = {2015},

date = {2015-12-24},

journal = {Nature},

volume = {528(7583)},

pages = {580-4},

abstract = {A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering. },

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{617,

title = {Engineering of Kuma030: a gliadin peptidase that rapidly degrades immunogenic gliadin peptides in gastric conditions.},

author = { Clancey Wolf and Justin B Siegel and Christine Tinberg and Alessandra Camarca and Carmen Gianfrani and Shirley Paski and Rongjin Guan and Gaetano T Montelione and David Baker and Ingrid S Pultz},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Wolf_JACS_2015.pdf},

doi = {10.1021/jacs.5b08325},

issn = {1520-5126},

year  = {2015},

date = {2015-09-01},

journal = {Journal of the American Chemical Society},

abstract = {Celiac disease is characterized by intestinal inflammation triggered by gliadin, a component of dietary gluten. Oral administration of proteases that can rapidly degrade gliadin in the gastric compartment has been proposed as a treatment for celiac disease; however, no protease has been shown to specifically reduce the immunogenic gliadin content, in gastric conditions, to below the threshold shown to be toxic for celiac patients. Here, we used the Rosetta Molecular Modeling Suite to redesign the active site of the acid-active gliadin endopeptidase KumaMax. The resulting protease, Kuma030, specifically recognizes tripeptide sequences that are found throughout the immunogenic regions of gliadin, as well as in homologous proteins in barley and rye. Indeed, treatment of gliadin with Kuma030 eliminates the ability of gliadin to stimulate a T cell response. Kuma030 is capable of degrading >99% of the immunogenic gliadin fraction in laboratory-simulated gastric digestions with minutes, to a level below the toxic threshold for celiac patients, suggesting great potential for this enzyme as an oral therapeutic for celiac disease.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{609,

title = {Structural plasticity of helical nanotubes based on coiled-coil assemblies.},

author = { E H Egelman and C. Xu and F. DiMaio and E Magnotti and C Modlin and X Yu and E Wright and D Baker and V P Conticello},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/structuralplasticity_Baker2015.pdf},

doi = {10.1016/j.str.2014.12.008},

issn = {1878-4186},

year  = {2015},

date = {2015-02-01},

journal = {Structure (London, England : 1993)},

volume = {23},

pages = {280-9},

abstract = {Numerous instances can be seen in evolution in which protein quaternary structures have diverged while the sequences of the building blocks have remained fairly conserved. However, the path through which such divergence has taken place is usually not known. We have designed two synthetic 29-residue α-helical peptides, based on the coiled-coil structural motif, that spontaneously self-assemble into helical nanotubes in vitro. Using electron cryomicroscopy with a newly available direct electron detection capability, we can achieve near-atomic resolution of these thin structures. We show how conservative changes of only one or two amino acids result in dramatic changes in quaternary structure, in which the assemblies can be switched between two very different forms. This system provides a framework for understanding how small sequence changes in evolution can translate into very large changes in supramolecular structure, a phenomenon that may have significant implications for the de novo design of synthetic peptide assemblies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{619,

title = {Computational design of a red fluorophore ligase for site-specific protein labeling in living cells.},

author = { Daniel S Liu and Lucas G Niv'on and Florian Richter and Peter J Goldman and Thomas J Deerinck and Jennifer Z Yao and Douglas Richardson and William S Phipps and Anne Z Ye and Mark H Ellisman and Catherine L Drennan and David Baker and Alice Y Ting},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Liu_computational_PNAS_2014.pdf},

doi = {10.1073/pnas.1404736111},

issn = {1091-6490},

year  = {2014},

date = {2014-10-01},

journal = {Proceedings of the National Academy of Sciences of the United States of America},

volume = {111},

pages = {E4551-9},

abstract = {Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{533,

title = {Removing T-cell epitopes with computational protein design.},

author = { Chris King and Esteban N Garza and Ronit Mazor and Jonathan L Linehan and Ira Pastan and Marion Pepper and David Baker},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/King_PNAS_2014A.pdf},

doi = {10.1073/pnas.1321126111},

issn = {1091-6490},

year  = {2014},

date = {2014-05-01},

journal = {Proceedings of the National Academy of Sciences of the United States of America},

abstract = {Immune responses can make protein therapeutics ineffective or even dangerous. We describe a general computational protein design method for reducing immunogenicity by eliminating known and predicted T-cell epitopes and maximizing the content of human peptide sequences without disrupting protein structure and function. We show that the method recapitulates previous experimental results on immunogenicity reduction, and we use it to disrupt T-cell epitopes in GFP and Pseudomonas exotoxin A without disrupting function.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{510,

title = {Amyloid β peptide cleavage by kallikrein 7 attenuates fibril growth and rescues neurons from Aβ-mediated toxicity in vitro.},

author = { Tyler D Shropshire and Jack Reifert and Sridharan Rajagopalan and David Baker and Stuart C Feinstein and Patrick S Daugherty},

doi = {10.1515/hsz-2013-0230},

issn = {1437-4315},

year  = {2014},

date = {2014-01-01},

journal = {Biological chemistry},

volume = {395},

pages = {109-18},

abstract = {Abstract The gradual accumulation and assembly of β-amyloid (Aβ) peptide into neuritic plaques is a major pathological hallmark of Alzheimer disease (AD). Proteolytic degradation of Aβ is an important clearance mechanism under normal circumstances, and it has been found to be compromised in those with AD. Here, the extended substrate specificity and Aβ-degrading capacity of kallikrein 7 (KLK7), a serine protease with a unique chymotrypsin-like specificity, was characterized. Preferred peptide substrates of KLK7 identified using a bacterial display substrate library were found to exhibit a consensus motif of RXΦ(Y/F)textdownarrow(Y/F)textdownarrow(S/A/G/T) or RXΦ(Y/F)textdownarrow(S/T/A) (Φ=hydrophobic), which is remarkably similar to the hydrophobic core motif of Aβ (K16L17V18F19F20 A21) that is largely responsible for aggregation propensity. KLK7 was found to cleave after both Phe residues within the core of Aβ42 in vitro, thereby inhibiting Aβ fibril formation and promoting the degradation of preformed fibrils. Finally, the treatment of Aβ oligomer preparations with KLK7, but not inactive pro-KLK7, significantly reduced Aβ42-mediated toxicity to rat hippocampal neurons to the same extent as the known Aβ-degrading protease insulin-degrading enzyme (IDE). Taken together, these results indicate that KLK7 possesses an Aβ-degrading capacity that can ameliorate the toxic effects of the aggregated peptide in vitro.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{618,

title = {VaxCelerate II: rapid development of a self-assembling vaccine for Lassa fever.},

author = { Pierre Leblanc and Leonard Moise and Cybelle Luza and Kanawat Chantaralawan and Lynchy Lezeau and Jianping Yuan and Mary Field and Daniel Richer and Christine Boyle and William D Martin and Jordan B Fishman and Eric A Berg and David Baker and Brandon Zeigler and Dale E Mais and William Taylor and Russell Coleman and H Shaw Warren and Jeffrey A Gelfand and Anne S De Groot and Timothy Brauns and Mark C Poznansky},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Leblanc_HumanVI_2014.pdf},

doi = {10.4161/hv.34413},

issn = {2164-554X},

year  = {2014},

date = {2014-00-01},

journal = {Human vaccines & immunotherapeutics},

volume = {10},

pages = {3022-38},

abstract = {Development of effective vaccines against emerging infectious diseases (EID) can take as much or more than a decade to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. Lassa is a prototypical emerging infectious disease endemic to West Africa for which no successful vaccine is available. We established the VaxCelerate Consortium to address the need for more rapid vaccine development by creating a platform capable of generating and pre-clinically testing a new vaccine against specific pathogen targets in less than 120 d A self-assembling vaccine is at the core of the approach. It consists of a fusion protein composed of the immunostimulatory Mycobacterium tuberculosis heat shock protein 70 (MtbHSP70) and the biotin binding protein, avidin. Mixing the resulting protein (MAV) with biotinylated pathogen-specific immunogenic peptides yields a self-assembled vaccine (SAV). To meet the time constraint imposed on this project, we used a distributed R&D model involving experts in the fields of protein engineering and production, bioinformatics, peptide synthesis/design and GMP/GLP manufacturing and testing standards. SAV immunogenicity was first tested using H1N1 influenza specific peptides and the entire VaxCelerate process was then tested in a mock live-fire exercise targeting Lassa fever virus. We demonstrated that the Lassa fever vaccine induced significantly increased class II peptide specific interferon-γ CD4(+) T cell responses in HLA-DR3 transgenic mice compared to peptide or MAV alone controls. We thereby demonstrated that our SAV in combination with a distributed development model may facilitate accelerated regulatory review by using an identical design for each vaccine and by applying safety and efficacy assessment tools that are more relevant to human vaccine responses than current animal models.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{605,

title = {Remodeling a beta-peptide bundle},

author = { MA Molski and JL Goodman and FC Chou and D Baker and R Das and A Schepartz},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/remodelingabeta_Baker2013.pdf},

doi = {10.1039/c2sc21117c},

issn = {2041-6520},

year  = {2013},

date = {2013-00-01},

journal = {Chemical Science},

volume = {4},

pages = {319-324},

abstract = {Natural biopolymers fold with fidelity, burying diverse side chains into well-packed cores and protecting their backbones from solvent. Certain beta-peptide oligomers assemble into bundles of defined octameric stoichiometry that resemble natural proteins in many respects. These beta-peptide bundles are thermostable, fold cooperatively, exchange interior amide N-H protons slowly, exclude hydrophobic dyes, and can be characterized at high resolution using X-ray crystallography - just like many proteins found in nature. But unlike natural proteins, all octameric beta-peptide bundles contain a sequence-uniform hydrophobic core composed of 32 leucine side chains. Here we apply rational design principles, including the Rosetta computational design methodology, to introduce sequence diversity into the bundle core while retaining the characteristic beta-peptide bundle fold. Using circular dichroism spectroscopy and analytical ultracentrifugation, we confirmed the prediction that an octameric bundle still assembles upon a major remodelling of its core: the mutation of sixteen core beta-homo-leucine side chains into sixteen beta-homo-phenylalanine side chains. Nevertheless, the bundle containing a partially beta-homo-phenylalanine core poorly protects interior amide protons from exchange, suggesting molten-globule-like properties. We further improve stability by the incorporation of eight beta-homo-pentafluorophenyalanine side chains, giving an assembly with amide protection factors comparable to prior well-structured bundles. By demonstrating that their cores tolerate significant sequence variation, the beta-peptide bundles reported here represent a starting point for the "bottom-up" construction of beta-peptide assemblies possessing both structure and sophisticated function.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{479,

title = {Computational Design of an α-gliadin Peptidase},

author = { Sydney R Gordon and Elizabeth J Stanley and Sarah Wolf and Angus Toland and Sean J Wu and Daniel Hadidi and Jeremy H Mills and David Baker and Ingrid Swanson Pultz and Justin B Siegel},

url = {https://www.bakerlab.org/wp-content/uploads/2015/12/Gordon12E.pdf

http://www.ncbi.nlm.nih.gov/pubmed/23153249},

doi = {10.1021/ja3094795},

issn = {1520-5126},

year  = {2012},

date = {2012-12-01},

journal = {Journal of the American Chemical Society},

volume = {134},

pages = {20513-20},

abstract = {The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics. Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease. The engineered enzyme exhibits a k(cat)/K(M) of 568 M(-1) s(-1), representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides. The computationally engineered enzyme is resistant to proteolysis by digestive proteases and degrades over 95% of an immunogenic peptide implicated in celiac disease in under an hour. Thus, through identification of a natural enzyme with the pre-existing qualities relevant to an ultimate goal and redefinition of its substrate specificity using computational modeling, we were able to generate an enzyme with potential as a therapeutic for celiac disease.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{455,

title = {Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases.},

author = { Sarah Baxter and Abigail R Lambert and Ryan Kuhar and Jordan Jarjour and Nadia Kulshina and Fabio Parmeggiani and Patrick Danaher and Jacob Gano and David Baker and Barry L Stoddard and Andrew M Scharenberg},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Baxter_NuclAcRes_2012.pdf},

issn = {1362-4962},

year  = {2012},

date = {2012-09-01},

journal = {Nucleic acids research},

volume = {40},

pages = {7985-8000},

abstract = {Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40-70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ~70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{600,

title = {The dynamic disulphide relay of quiescin sulphydryl oxidase},

author = { Assaf Alon and Iris Grossman and Yair Gat and Vamsi K Kodali and Frank DiMaio and Tevie Mehlman and Gilad Haran and David Baker and Colin Thorpe and Deborah Fass},

url = {https://www.bakerlab.org/wp-content/uploads/2016/01/thedynamicdisulphide_Baker2012.pdf},

doi = {10.1038/nature11267},

issn = {1476-4687},

year  = {2012},

date = {2012-08-01},

journal = {Nature},

volume = {488},

pages = {414-8},

abstract = {Protein stability, assembly, localization and regulation often depend on the formation of disulphide crosslinks between cysteine side chains. Enzymes known as sulphydryl oxidases catalyse de novo disulphide formation and initiate intra- and intermolecular dithiol/disulphide relays to deliver the disulphides to substrate proteins. Quiescin sulphydryl oxidase (QSOX) is a unique, multi-domain disulphide catalyst that is localized primarily to the Golgi apparatus and secreted fluids and has attracted attention owing to its overproduction in tumours. In addition to its physiological importance, QSOX is a mechanistically intriguing enzyme, encompassing functions typically carried out by a series of proteins in other disulphide-formation pathways. How disulphides are relayed through the multiple redox-active sites of QSOX and whether there is a functional benefit to concatenating these sites on a single polypeptide are open questions. Here we present the first crystal structure of an intact QSOX enzyme, derived from a trypanosome parasite. Notably, sequential sites in the disulphide relay were found more than 40 r A apart in this structure, too far for direct disulphide transfer. To resolve this puzzle, we trapped and crystallized an intermediate in the disulphide hand-off, which showed a 165textdegree domain rotation relative to the original structure, bringing the two active sites within disulphide-bonding distance. The comparable structure of a mammalian QSOX enzyme, also presented here, shows further biochemical features that facilitate disulphide transfer in metazoan orthologues. Finally, we quantified the contribution of concatenation to QSOX activity, providing general lessons for the understanding of multi-domain enzymes and the design of new catalytic relays.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{591,

title = {Improvement of a potential anthrax therapeutic by computational protein design},

author = { Sean J Wu and Christopher B Eiben and John H Carra and Ivan Huang and David Zong and Peixian Liu and Cindy T Wu and Jeff Nivala and Josef Dunbar and Tomas Huber and Jeffrey Senft and Rowena Schokman and Matthew D Smith and Jeremy H Mills and Arthur M Friedlander and David Baker and Justin B Siegel},

url = {https://www.bakerlab.org/wp-content/uploads/2018/06/J.-Biol.-Chem.-2011-Wu-32586-92.pdf

http://www.jbc.org/content/286/37/32586},

doi = {10.1074/jbc.M111.251041},

issn = {1083-351X},

year  = {2011},

date = {2011-09-01},

journal = {The Journal of Biological Chemistry},

volume = {286},

pages = {32586-92},

abstract = {Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 textdegreeC, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{401,

title = {Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation},

author = { Stuart A Sievers and John Karanicolas and Howard W Chang and Anni Zhao and Lin Jiang and Onofrio Zirafi and Jason T Stevens and Jan M"unch and David Baker and David Eisenberg},

doi = {10.1038/nature10154},

issn = {1476-4687},

year  = {2011},

date = {2011-06-01},

journal = {Nature},

abstract = {Many globular and natively disordered proteins can convert into amyloid fibrils. These fibrils are associated with numerous pathologies as well as with normal cellular functions, and frequently form during protein denaturation. Inhibitors of pathological amyloid fibril formation could be useful in the development of therapeutics, provided that the inhibitors were specific enough to avoid interfering with normal processes. Here we show that computer-aided, structure-based design can yield highly specific peptide inhibitors of amyloid formation. Using known atomic structures of segments of amyloid fibrils as templates, we have designed and characterized an all-d-amino-acid inhibitor of the fibril formation of the tau protein associated with Alzheimertextquoterights disease, and a non-natural l-amino-acid inhibitor of an amyloid fibril that enhances sexual transmission of human immunodeficiency virus. Our results indicate that peptides from structure-based designs can disrupt the fibril formation of full-length proteins, including those, such as tau protein, that lack fully ordered native structures. Because the inhibiting peptides have been designed on structures of dual-β-sheet textquoterightsteric zipperstextquoteright, the successful inhibition of amyloid fibril formation strengthens the hypothesis that amyloid spines contain steric zippers.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{357,

title = {Heterologous epitope-scaffold prime:boosting immuno-focuses B cell responses to the HIV-1 gp41 2F5 neutralization determinant},

author = { Javier Guenaga and Pia Dosenovic and Gilad Ofek and David Baker and William R Schief and Peter D Kwong and Gunilla B Karlsson Hedestam and Richard T Wyatt},

issn = {1932-6203},

year  = {2011},

date = {2011-00-01},

journal = {PloS one},

volume = {6},

pages = {e16074},

abstract = {The HIV-1 envelope glycoproteins (Env) gp120 and gp41 mediate entry and are the targets for neutralizing antibodies. Within gp41, a continuous epitope defined by the broadly neutralizing antibody 2F5, is one of the few conserved sites accessible to antibodies on the functional HIV Env spike. Recently, as an initial attempt at structure-guided design, we transplanted the 2F5 epitope onto several non-HIV acceptor scaffold proteins that we termed epitope scaffolds (ES). As immunogens, these ES proteins elicited antibodies with exquisite binding specificity matching that of the 2F5 antibody. These novel 2F5 epitope scaffolds presented us with the opportunity to test heterologous prime:boost immunization strategies to selectively boost antibody responses against the engrafted gp41 2F5 epitope. Such strategies might be employed to target conserved but poorly immunogenic sites on the HIV-1 Env, and, more generally, other structurally defined pathogen targets. Here, we assessed ES prime:boosting by measuring epitope specific serum antibody titers by ELISA and B cell responses by ELISpot analysis using both free 2F5 peptide and an unrelated ES protein as probes. We found that the heterologous ES prime:boosting immunization regimen elicits cross-reactive humoral responses to the structurally constrained 2F5 epitope target, and that incorporating a promiscuous T cell helper epitope in the immunogens resulted in higher antibody titers against the 2F5 graft, but did not result in virus neutralization. Interestingly, two epitope scaffolds (ES1 and ES2), which did not elicit a detectable 2F5 epitope-specific response on their own, boosted such responses when primed with the ES5. Together, these results indicate that heterologous ES prime:boost immunization regimens effectively focus the humoral immune response on the structurally defined and immunogen-conserved HIV-1 2F5 epitope.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{270,

title = {Elicitation of structure-specific antibodies by epitope scaffolds},

author = { Gilad Ofek and F Javier Guenaga and William R Schief and Jeff Skinner and David Baker and Richard Wyatt and Peter D Kwong},

issn = {1091-6490},

year  = {2010},

date = {2010-10-01},

journal = {Proceedings of the National Academy of Sciences of the United States of America},

volume = {107},

pages = {17880-7},

abstract = {Elicitation of antibodies against targets that are immunorecessive, cryptic, or transient in their native context has been a challenge for vaccine design. Here we demonstrate the elicitation of structure-specific antibodies against the HIV-1 gp41 epitope of the broadly neutralizing antibody 2F5. This conformationally flexible region of gp41 assumes mostly helical conformations but adopts a kinked, extended structure when bound by antibody 2F5. Computational techniques were employed to transplant the 2F5 epitope into select acceptor scaffolds. The resultant "2F5-epitope scaffolds" possessed nanomolar affinity for antibody 2F5 and a range of epitope flexibilities and antigenic specificities. Crystallographic characterization of the epitope scaffold with highest affinity and antigenic discrimination confirmed good to near perfect attainment of the target conformation for the gp41 molecular graft in free and 2F5-bound states, respectively. Animals immunized with 2F5-epitope scaffolds showed levels of graft-specific immune responses that correlated with graft flexibility (p~<~0.04), while antibody responses against the graft-as dissected residue-by-residue with alanine substitutions-resembled more closely those of 2F5 than sera elicited with flexible or cyclized peptides, a resemblance heightened by heterologous prime-boost. Lastly, crystal structures of a gp41 peptide in complex with monoclonal antibodies elicited by the 2F5-epitope scaffolds revealed that the elicited antibodies induce gp41 to assume its 2F5-recognized shape. Epitope scaffolds thus provide a means to elicit antibodies that recognize a predetermined target shape and sequence, even if that shape is transient in nature, and a means by which to dissect factors influencing such elicitation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{228,

title = {Computational design of epitope-scaffolds allows induction of antibodies specific for a poorly immunogenic HIV vaccine epitope},

author = { Bruno E Correia and Yih-En Andrew Ban and Margaret A Holmes and Hengyu Xu and Katharine Ellingson and Zane Kraft and Chris Carrico and Erica Boni and D Noah Sather and Camille Zenobia and Katherine Y Burke and Tyler Bradley-Hewitt and Jessica F Bruhn-Johannsen and Oleksandr Kalyuzhniy and David Baker and Roland K Strong and Leonidas Stamatatos and William R Schief},

issn = {1878-4186},

year  = {2010},

date = {2010-09-01},

journal = {Structure},

volume = {18},

pages = {1116-26},

abstract = {Broadly cross-reactive monoclonal antibodies define epitopes for vaccine development against HIV and other highly mutable viruses. Crystal structures are available for several such antibody-epitope complexes, but methods are needed to translate that structural information into immunogens that re-elicit similar antibodies. We describe a general computational method to design epitope-scaffolds in which contiguous structural epitopes are transplanted to scaffold proteins for conformational stabilization and immune presentation. Epitope-scaffolds designed for the poorly immunogenic but conserved HIV epitope 4E10 exhibited high epitope structural mimicry, bound with higher affinities to monoclonal antibody (mAb) 4E10 than the cognate peptide, and inhibited HIV neutralization by HIV+ sera. Rabbit immunization with an epitope-scaffold induced antibodies with structural specificity highly similar to mAb 4E10, an important advance toward elicitation of neutralizing activity. The results demonstrate that computationally designed epitope-scaffolds are valuable as structure-specific serological reagents and as immunogens to elicit antibodies with predetermined structural specificity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{267,

title = {High-resolution mapping of protein sequence-function relationships},

author = { Douglas M Fowler and Carlos L Araya and Sarel J Fleishman and Elizabeth H Kellogg and Jason J Stephany and David Baker and Stanley Fields},

issn = {1548-7105},

year  = {2010},

date = {2010-09-01},

journal = {Nature methods},

volume = {7},

pages = {741-6},

abstract = {We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, moderate selection for activity and high-throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position had unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{271,

title = {Feature space resampling for protein conformational search},

author = { Ben Blum and Michael I Jordan and David Baker},

issn = {1097-0134},

year  = {2010},

date = {2010-05-01},

journal = {Proteins},

volume = {78},

pages = {1583-93},

abstract = {De novo protein structure prediction requires location of the lowest energy state of the polypeptide chain among a vast set of possible conformations. Powerful approaches include conformational space annealing, in which search progressively focuses on the most promising regions of conformational space, and genetic algorithms, in which features of the best conformations thus far identified are recombined. We describe a new approach that combines the strengths of these two approaches. Protein conformations are projected onto a discrete feature space which includes backbone torsion angles, secondary structure, and beta pairings. For each of these there is one "native" value: the one found in the native structure. We begin with a large number of conformations generated in independent Monte Carlo structure prediction trajectories from Rosetta. Native values for each feature are predicted from the frequencies of feature value occurrences and the energy distribution in conformations containing them. A second round of structure prediction trajectories are then guided by the predicted native feature distributions. We show that native features can be predicted at much higher than background rates, and that using the predicted feature distributions improves structure prediction in a benchmark of 28 proteins. The advantages of our approach are that features from many different input structures can be combined simultaneously without producing atomic clashes or otherwise physically inviable models, and that the features being recombined have a relatively high chance of being correct.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{582,

title = {Insights from the crystal structure of the sixth BRCT domain of topoisomerase IIbeta binding protein 1},

author = { Charles Chung Yun Leung and Elizabeth Kellogg and Anja Kuhnert and Frank H"anel and David Baker and J N Mark Glover},

doi = {10.1002/pro.290},

issn = {1469-896X},

year  = {2010},

date = {2010-01-01},

journal = {Protein science : a publication of the Protein Society},

volume = {19},

pages = {162-7},

abstract = {Topoisomerase IIbeta binding protein 1 (TopBP1) is a major player in the DNA damage response and interacts with a number of protein partners via its eight BRCA1 carboxy-terminal (BRCT) domains. In particular, the sixth BRCT domain of TopBP1 has been implicated in binding to the phosphorylated transcription factor, E2F1, and poly(ADP-ribose) polymerase 1 (PARP-1), where the latter interaction is responsible for the poly(ADP-ribosyl)ation of TopBP1. To gain a better understanding of the nature of TopBP1 BRCT6 interactions, we solved the crystal structure of BRCT6 to 1.34 A. The crystal structure reveals a degenerate phospho-peptide binding pocket and lacks conserved hydrophobic residues involved in packing of tandem BRCT repeats, which, together with results from phospho-peptide binding studies, strongly suggest that TopBP1 BRCT6 independently does not function as a phospho-peptide binding domain. We further provide insight into poly(ADP-ribose) binding and sites of potential modification by PARP-1.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{219,

title = {A new twist in TCR diversity revealed by a forbidden alphabeta TCR},

author = { Christine McBeth and Audrey Seamons and Juan C Pizarro and Sarel J Fleishman and David Baker and Tanja Kortemme and Joan M Goverman and Roland K Strong},

issn = {1089-8638},

year  = {2008},

date = {2008-02-01},

journal = {Journal of molecular biology},

volume = {375},

pages = {1306-19},

abstract = {We report crystal structures of a negatively selected T cell receptor (TCR) that recognizes two I-A(u)-restricted myelin basic protein peptides and one of its peptide/major histocompatibility complex (pMHC) ligands. Unusual complementarity-determining region (CDR) structural features revealed by our analyses identify a previously unrecognized mechanism by which the highly variable CDR3 regions define ligand specificity. In addition to the pMHC contact residues contributed by CDR3, the CDR3 residues buried deep within the V alpha/V beta interface exert indirect effects on recognition by influencing the V alpha/V beta interdomain angle. This phenomenon represents an additional mechanism for increasing the potential diversity of the TCR repertoire. Both the direct and indirect effects exerted by CDR residues can impact global TCR/MHC docking. Analysis of the available TCR structures in light of these results highlights the significance of the V alpha/V beta interdomain angle in determining specificity and indicates that TCR/pMHC interface features do not distinguish autoimmune from non-autoimmune class II-restricted TCRs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{121,

title = {Prediction of the structure of symmetrical protein assemblies},

author = { Ingemar Andr'e and Philip Bradley and Chu Wang and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/08/André07A.pdf},

issn = {0027-8424},

year  = {2007},

date = {2007-11-01},

journal = {Proceedings of the National Academy of Sciences of the United States of America},

volume = {104},

pages = {17656-61},

abstract = {Biological supramolecular systems are commonly built up by the self-assembly of identical protein subunits to produce symmetrical oligomers with cyclical, icosahedral, or helical symmetry that play roles in processes ranging from allosteric control and molecular transport to motor action. The large size of these systems often makes them difficult to structurally characterize using experimental techniques. We have developed a computational protocol to predict the structure of symmetrical protein assemblies based on the structure of a single subunit. The method carries out simultaneous optimization of backbone, side chain, and rigid-body degrees of freedom, while restricting the search space to symmetrical conformations. Using this protocol, we can reconstruct, starting from the structure of a single subunit, the structure of cyclic oligomers and the icosahedral virus capsid of satellite panicum virus using a rigid backbone approximation. We predict the oligomeric state of EscJ from the type III secretion system both in its proposed cyclical and crystallized helical form. Finally, we show that the method can recapitulate the structure of an amyloid-like fibril formed by the peptide NNQQNY from the yeast prion protein Sup35 starting from the amino acid sequence alone and searching the complete space of backbone, side chain, and rigid-body degrees of freedom.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{114,

title = {Cooperative hydrogen bonding in amyloid formation},

author = { Kiril Tsemekhman and Lukasz Goldschmidt and David Eisenberg and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/08/tsemekhman07A.pdf},

issn = {0961-8368},

year  = {2007},

date = {2007-04-01},

journal = {Protein science},

volume = {16},

pages = {761-4},

abstract = {Amyloid diseases, including Alzheimertextquoterights and prion diseases, are each associated with unbranched protein fibrils. Each fibril is made of a particular protein, yet they share common properties. One such property is nucleation-dependent fibril growth. Monomers of amyloid-forming proteins can remain in dissolved form for long periods, before rapidly assembly into fibrils. The lag before growth has been attributed to slow kinetics of formation of a nucleus, on which other molecules can deposit to form the fibril. We have explored the energetics of fibril formation, based on the known molecular structure of a fibril-forming peptide from the yeast prion, Sup35, using both classical and quantum (density functional theory) methods. We find that the energetics of fibril formation for the first three layers are cooperative using both methods. This cooperativity is consistent with the observation that formation of amyloid fibrils involves slow nucleation and faster growth.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{111,

title = {The highly cooperative folding of small naturally occurring proteins is likely the result of natural selection},

author = { Alexander L Watters and Pritilekha Deka and Colin Corrent and David Callender and Gabriele Varani and Tobin Sosnick and David Baker},

issn = {0092-8674},

year  = {2007},

date = {2007-02-01},

journal = {Cell},

volume = {128},

pages = {613-24},

abstract = {To illuminate the evolutionary pressure acting on the folding free energy landscapes of naturally occurring proteins, we have systematically characterized the folding free energy landscape of Top7, a computationally designed protein lacking an evolutionary history. Stopped-flow kinetics, circular dichroism, and NMR experiments reveal that there are at least three distinct phases in the folding of Top7, that a nonnative conformation is stable at equilibrium, and that multiple fragments of Top7 are stable in isolation. These results indicate that the folding of Top7 is significantly less cooperative than the folding of similarly sized naturally occurring proteins, suggesting that the cooperative folding and smooth free energy landscapes observed for small naturally occurring proteins are not general properties of polypeptide chains that fold to unique stable structures but are instead a product of natural selection.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{293,

title = {Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs},

author = { Amy E Palmer and Marta Giacomello and Tanja Kortemme and S Andrew Hires and Varda Lev-Ram and David Baker and Roger Y Tsien},

url = {https://www.bakerlab.org/wp-content/uploads/2016/08/palmer06A.pdf},

issn = {1074-5521},

year  = {2006},

date = {2006-05-01},

journal = {Chemistry & biology},

volume = {13},

pages = {521-30},

abstract = {The binding interface of calmodulin and a calmodulin binding peptide were reengineered by computationally designing complementary bumps and holes. This redesign led to the development of sensitive and specific pairs of mutant proteins used to sense Ca(2+) in a second generation of genetically encoded Ca(2+) indicators (cameleons). These cameleons are no longer perturbed by large excesses of native calmodulin, and they display Ca(2+) sensitivities tuned over a 100-fold range (0.6-160 microM). Incorporation of circularly permuted Venus in place of Citrine results in a 3- to 5-fold increase in the dynamic range. These redesigned cameleons show significant improvements over previous versions in the ability to monitor Ca(2+) in the cytoplasm as well as distinct subcellular localizations, such as the plasma membrane of neurons and the mitochondria.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{162,

title = {Recapitulation and design of protein binding peptide structures and sequences},

author = { Vanita D Sood and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/08/sood06A.pdf},

issn = {0022-2836},

year  = {2006},

date = {2006-03-01},

journal = {Journal of molecular biology},

volume = {357},

pages = {917-27},

abstract = {An important objective of computational protein design is the generation of high affinity peptide inhibitors of protein-peptide interactions, both as a precursor to the development of therapeutics aimed at disrupting disease causing complexes, and as a tool to aid investigators in understanding the role of specific complexes in the cell. We have developed a computational approach to increase the affinity of a protein-peptide complex by designing N or C-terminal extensions which interact with the protein outside the canonical peptide binding pocket. In a first in silico test, we show that by simultaneously optimizing the sequence and structure of three to nine residue peptide extensions starting from short (1-6 residue) peptide stubs in the binding pocket of a peptide binding protein, the approach can recover both the conformations and the sequences of known binding peptides. Comparison with phage display and other experimental data suggests that the peptide extension approach recapitulates naturally occurring peptide binding specificity better than fixed backbone design, and that it should be useful for predicting peptide binding specificities from crystal structures. We then experimentally test the approach by designing extensions for p53 and dystroglycan-based peptides predicted to bind with increased affinity to the Mdm2 oncoprotein and to dystrophin, respectively. The measured increases in affinity are modest, revealing some limitations of the method. Based on these in silico and experimental results, we discuss future applications of the approach to the prediction and design of protein-peptide interactions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{163,

title = {The 3D profile method for identifying fibril-forming segments of proteins},

author = { Michael J Thompson and Stuart A Sievers and John Karanicolas and Magdalena I Ivanova and David Baker and David Eisenberg},

url = {https://www.bakerlab.org/wp-content/uploads/2016/08/thompson06A.pdf},

issn = {0027-8424},

year  = {2006},

date = {2006-03-01},

journal = {Proceedings of the National Academy of Sciences of the United States of America},

volume = {103},

pages = {4074-8},

abstract = {Based on the crystal structure of the cross-beta spine formed by the peptide NNQQNY, we have developed a computational approach for identifying those segments of amyloidogenic proteins that themselves can form amyloid-like fibrils. The approach builds on experiments showing that hexapeptides are sufficient for forming amyloid-like fibrils. Each six-residue peptide of a protein of interest is mapped onto an ensemble of templates, or 3D profile, generated from the crystal structure of the peptide NNQQNY by small displacements of one of the two intermeshed beta-sheets relative to the other. The energy of each mapping of a sequence to the profile is evaluated by using ROSETTADESIGN, and the lowest energy match for a given peptide to the template library is taken as the putative prediction. If the energy of the putative prediction is lower than a threshold value, a prediction of fibril formation is made. This method can reach an accuracy of approximately 80% with a P value of approximately 10(-12) when a conservative energy threshold is used to separate peptides that form fibrils from those that do not. We see enrichment for positive predictions in a set of fibril-forming segments of amyloid proteins, and we illustrate the method with applications to proteins of interest in amyloid research.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{177,

title = {Modeling structurally variable regions in homologous proteins with rosetta},

author = { Carol A Rohl and Charlie E M Strauss and Dylan Chivian and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/06/rohl04A.pdf},

issn = {1097-0134},

year  = {2004},

date = {2004-05-01},

journal = {Proteins},

volume = {55},

pages = {656-77},

abstract = {A major limitation of current comparative modeling methods is the accuracy with which regions that are structurally divergent from homologues of known structure can be modeled. Because structural differences between homologous proteins are responsible for variations in protein function and specificity, the ability to model these differences has important functional consequences. Although existing methods can provide reasonably accurate models of short loop regions, modeling longer structurally divergent regions is an unsolved problem. Here we describe a method based on the de novo structure prediction algorithm, Rosetta, for predicting conformations of structurally divergent regions in comparative models. Initial conformations for short segments are selected from the protein structure database, whereas longer segments are built up by using three- and nine-residue fragments drawn from the database and combined by using the Rosetta algorithm. A gap closure term in the potential in combination with modified Newtontextquoterights method for gradient descent minimization is used to ensure continuity of the peptide backbone. Conformations of variable regions are refined in the context of a fixed template structure using Monte Carlo minimization together with rapid repacking of side-chains to iteratively optimize backbone torsion angles and side-chain rotamers. For short loops, mean accuracies of 0.69, 1.45, and 3.62 A are obtained for 4, 8, and 12 residue loops, respectively. In addition, the method can provide reasonable models of conformations of longer protein segments: predicted conformations of 3A root-mean-square deviation or better were obtained for 5 of 10 examples of segments ranging from 13 to 34 residues. In combination with a sequence alignment algorithm, this method generates complete, ungapped models of protein structures, including regions both similar to and divergent from a homologous structure. This combined method was used to make predictions for 28 protein domains in the Critical Assessment of Protein Structure 4 (CASP 4) and 59 domains in CASP 5, where the method ranked highly among comparative modeling and fold recognition methods. Model accuracy in these blind predictions is dominated by alignment quality, but in the context of accurate alignments, long protein segments can be accurately modeled. Notably, the method correctly predicted the local structure of a 39-residue insertion into a TIM barrel in CASP 5 target T0186.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{574,

title = {Exploring folding free energy landscapes using computational protein design},

author = { Brian Kuhlman and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/06/exploringfoldingfreeenergy_Baker2004.pdf},

doi = {10.1016/j.sbi.2004.01.002},

issn = {0959-440X},

year  = {2004},

date = {2004-02-01},

journal = {Current opinion in structural biology},

volume = {14},

pages = {89-95},

abstract = {Recent advances in computational protein design have allowed exciting new insights into the sequence dependence of protein folding free energy landscapes. Whereas most previous studies have examined the sequence dependence of protein stability and folding kinetics by characterizing naturally occurring proteins and variants of these proteins that contain a small number of mutations, it is now possible to generate and characterize computationally designed proteins that differ significantly from naturally occurring proteins in sequence and/or structure. These computer-generated proteins provide insights into the determinants of protein structure, stability and folding, and make it possible to disentangle the properties of proteins that are the consequence of natural selection from those that reflect the fundamental physical chemistry of polypeptide chains.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{73,

title = {Efficient minimization of angle-dependent potentials for polypeptides in internal coordinates},

author = { William J Wedemeyer and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/06/wedemeyer03A.pdf},

issn = {1097-0134},

year  = {2003},

date = {2003-11-01},

journal = {Proteins},

volume = {53},

pages = {262-72},

abstract = {Angular potentials play an important role in the refinement of protein structures through angle-dependent restraints (e.g., those determined by cross-correlated relaxations, residual dipolar couplings, and hydrogen bonds). Analytic derivatives of such angular potentials with respect to the dihedral angles of proteins would be useful for optimizing such restraints and other types of angular potentials (i.e., such as we are now introducing into protein structure prediction) but have not been described. In this article, analytic derivatives are calculated for four types of angular potentials and integrated with the efficient recursive derivative calculation methods of Go and coworkers. The formulas are implemented in publicly available software and illustrated by refining a low-resolution protein structure with idealized vector-angle, dipolar-coupling, and hydrogen-bond restraints. The method is now being used routinely to optimize hydrogen-bonding potentials in ROSETTA.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{316,

title = {Profile-profile comparisons by COMPASS predict intricate homologies between protein families},

author = { Ruslan I Sadreyev and David Baker and Nick V Grishin},

url = {https://www.bakerlab.org/wp-content/uploads/2016/06/sadreyev03A.pdf},

issn = {0961-8368},

year  = {2003},

date = {2003-10-01},

journal = {Protein science},

volume = {12},

pages = {2262-72},

abstract = {Recently we proposed a novel method of alignment-alignment comparison, COMPASS (the tool for COmparison of Multiple Protein Alignments with Assessment of Statistical Significance). Here we present several examples of the relations between PFAM protein families that were detected by COMPASS and that lead to the predictions of presently unresolved protein structures. We discuss relatively straightforward COMPASS predictions that are new and interesting to us, and that would require a substantial time and effort to justify even for a skilled PSI-BLAST user. All of the presented COMPASS hits are independently confirmed by other methods, including the ab initio structure-prediction method ROSETTA. The tertiary structure predictions made by ROSETTA proved to be useful for improving sequence-derived alignments, because they are based on a reasonable folding of the polypeptide chain rather than on the information from sequence databases. The ability of COMPASS to predict new relations within the PFAM database indicates the high sensitivity of COMPASS searches and substantiates its potential value for the discovery of previously unknown similarities between protein families.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{84,

title = {Contact order revisited: influence of protein size on the folding rate},

author = { Dmitry N Ivankov and Sergiy O Garbuzynskiy and Eric Alm and Kevin W Plaxco and David Baker and Alexei V Finkelstein},

url = {https://www.bakerlab.org/wp-content/uploads/2016/06/ivankov03A.pdf},

issn = {0961-8368},

year  = {2003},

date = {2003-09-01},

journal = {Protein science},

volume = {12},

pages = {2057-62},

abstract = {Guided by the recent success of empirical model predicting the folding rates of small two-state folding proteins from the relative contact order (CO) of their native structures, by a theoretical model of protein folding that predicts that logarithm of the folding rate decreases with the protein chain length L as L(2/3), and by the finding that the folding rates of multistate folding proteins strongly correlate with their sizes and have very bad correlation with CO, we reexamined the dependence of folding rate on CO and L in attempt to find a structural parameter that determines folding rates for the totality of proteins. We show that the Abs_CO = CO x L, is able to predict rather accurately folding rates for both two-state and multistate folding proteins, as well as short peptides, and that this Abs_CO scales with the protein chain length as L(0.70 +/- 0.07) for the totality of studied single-domain proteins and peptides.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{76,

title = {Low free energy cost of very long loop insertions in proteins},

author = { Michelle Scalley-Kim and Philippe Minard and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/06/scalley-kim03A.pdf},

issn = {0961-8368},

year  = {2003},

date = {2003-02-01},

journal = {Protein science},

volume = {12},

pages = {197-206},

abstract = {Long insertions into a loop of a folded host protein are expected to have destabilizing effects because of the entropic cost associated with loop closure unless the inserted sequence adopts a folded structure with amino- and carboxy-termini in close proximity. A loop entropy reduction screen based on this concept was used in an attempt to retrieve folded sequences from random sequence libraries. A library of long random sequences was inserted into a loop of the SH2 domain, displayed on the surface of M13 phage, and the inserted sequences that did not disrupt SH2 function were retrieved by panning using beads coated with a phosphotyrosine containing SH2 peptide ligand. Two sequences of a library of 2 x 10(8) sequences were isolated after multiple rounds of panning, and were found to have recovery levels similar to the wild-type SH2 domain and to be relatively intolerant to further mutation in PCR mutagenesis experiments. Surprisingly, although these inserted sequences exhibited little nonrandom structure, they do not significantly destabilize the host SH2 domain. Additional insertion variants recovered at lower levels in the panning experiments were also found to have a minimal effect on the stability and peptide-binding function of the SH2 domain. The additional level of selection present in the panning experiments is likely to involve in vivo folding and assembly, as there was a rough correlation between recovery levels in the phage-panning experiments and protein solubility. The finding that loop insertions of 60-80 amino acids have minimal effects on SH2 domain stability suggests that the free energy cost of inserting long loops may be considerably less than polymer theory estimates based on the entropic cost of loop closure, and, hence, that loop insertion may have provided an evolutionary route to multidomain protein structures.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{66,

title = {Mechanisms of protein folding.},

author = { V Grantcharova and E J Alm and David Baker and A L Horwich},

issn = {0959-440X},

year  = {2001},

date = {2001-02-01},

journal = {Current opinion in structural biology},

volume = {11},

pages = {70-82},

abstract = {The strong correlation between protein folding rates and the contact order suggests that folding rates are largely determined by the topology of the native structure. However, for a given topology, there may be several possible low free energy paths to the native state and the path that is chosen (the lowest free energy path) may depend on differences in interaction energies and local free energies of ordering in different parts of the structure. For larger proteins whose folding is assisted by chaperones, such as the Escherichia coli chaperonin GroEL, advances have been made in understanding both the aspects of an unfolded protein that GroEL recognizes and the mode of binding to the chaperonin. The possibility that GroEL can remove non-native proteins from kinetic traps by unfolding them either during polypeptide binding to the chaperonin or during the subsequent ATP-dependent formation of folding-active complexes with the co-chaperonin GroES has also been explored.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{193,

title = {De novo protein structure determination using sparse NMR data.},

author = { P M Bowers and C E Strauss and David Baker},

issn = {0925-2738},

year  = {2000},

date = {2000-12-01},

journal = {Journal of biomolecular NMR},

volume = {18},

pages = {311-8},

abstract = {We describe a method for generating moderate to high-resolution protein structures using limited NMR data combined with the ab initio protein structure prediction method Rosetta. Peptide fragments are selected from proteins of known structure based on sequence similarity and consistency with chemical shift and NOE data. Models are built from these fragments by minimizing an energy function that favors hydrophobic burial, strand pairing, and satisfaction of NOE constraints. Models generated using this procedure with approximately 1 NOE constraint per residue are in some cases closer to the corresponding X-ray structures than the published NMR solution structures. The method requires only the sparse constraints available during initial stages of NMR structure determination, and thus holds promise for increasing the speed with which protein solution structures can be determined.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{194,

title = {HMMSTR: a hidden Markov model for local sequence-structure correlations in proteins},

author = { C Bystroff and V Thorsson and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/06/bystroff00A.pdf},

issn = {0022-2836},

year  = {2000},

date = {2000-08-01},

journal = {Journal of molecular biology},

volume = {301},

pages = {173-90},

abstract = {We describe a hidden Markov model, HMMSTR, for general protein sequence based on the I-sites library of sequence-structure motifs. Unlike the linear hidden Markov models used to model individual protein families, HMMSTR has a highly branched topology and captures recurrent local features of protein sequences and structures that transcend protein family boundaries. The model extends the I-sites library by describing the adjacencies of different sequence-structure motifs as observed in the protein database and, by representing overlapping motifs in a much more compact form, achieves a great reduction in parameters. The HMM attributes a considerably higher probability to coding sequence than does an equivalent dipeptide model, predicts secondary structure with an accuracy of 74.3 %, backbone torsion angles better than any previously reported method and the structural context of beta strands and turns with an accuracy that should be useful for tertiary structure prediction.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{192,

title = {A surprising simplicity to protein folding},

author = { David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/06/baker00A.pdf},

issn = {0028-0836},

year  = {2000},

date = {2000-05-01},

journal = {Nature},

volume = {405},

pages = {39-42},

abstract = {The polypeptide chains that make up proteins have thousands of atoms and hence millions of possible inter-atomic interactions. It might be supposed that the resulting complexity would make prediction of protein structure and protein-folding mechanisms nearly impossible. But the fundamental physics underlying folding may be much simpler than this complexity would lead us to expect folding rates and mechanisms appear to be largely determined by the topology of the native (folded) state, and new methods have shown great promise in predicting protein-folding mechanisms and the three-dimensional structures of proteins.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{42,

title = {Structural transitions in the protein L denatured state ensemble},

author = { M L Scalley and S Nauli and S T Gladwin and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/06/scalley99A.pdf},

issn = {0006-2960},

year  = {1999},

date = {1999-11-01},

journal = {Biochemistry},

volume = {38},

pages = {15927-35},

abstract = {We use a broad array of biophysical methods to probe the extent of structure and time scale of structural transitions in the protein L denatured state ensemble. Measurement of amide proton exchange protection during the first several milliseconds following initiation of refolding in 0.4 M sodium sulfate revealed weak protection in the first beta-hairpin and helix. A tryptophan residue was introduced into the first beta-hairpin to probe the extent of structure formation in this part of the protein; the intrinsic fluorescence of this tryptophan was found to deviate from that expected given its local sequence context in 2-3 M guanidine, suggesting some partial ordering of this region in the unfolded state ensemble. To further probe this partial ordering, dansyl groups were introduced via cysteine residues at three sites in the protein. It was found that fluorescence energy transfer from the introduced tryptophan to the dansyl groups decreased dramatically upon unfolding. Stopped-flow fluorescence studies showed that the recovery of dansyl fluorescence upon refolding occurred on a submillisecond time scale. To probe the interactions responsible for the residual structure observed in the denatured state ensemble, the conformation of a peptide corresponding to the first beta-hairpin and helix of protein L was studied using circular dichroism spectroscopy and compared to that of full-length protein L and previously characterized peptides corresponding to the isolated helix and second beta-hairpin.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}