Computational design of an enzyme catalyst for a stereoselective bimolecular Diels-Alder reaction

 Siegel and Zanghellini et al, Science, 329, 309-313. (2010)

Structure of a designed Diels-Alderase. (A) Surface view of the design model (DA_20_00, green) bound to the substrates (diene and dienophile, purple). The catalytic residues making the designed hydrogen bonds are depicted as sticks. (B) Overlay of the design model (DA_20_00,brown) and crystal structure of DA_20_00_A74I (green).  The crystal structure shows that the design was within atomic level accuracy (0.5A all-atom RMSD) 

Structure prediction problems solved by Foldit players

Cooper, Khatib et al, Nature, 466, 756-60. (2010)

Examples of blind structure prediction problems in which players were successfully able to improve structures. Native structures are shown in blue, starting puzzles in red, and top scoring Foldit predictions in green.

(a) The red starting puzzle had a register shift and the top scoring green Foldit prediction correctly flips and slides the beta strand.

(b) On the same structure as above, Foldit players correctly buried an exposed Isoleucine in the loop on the bottom right by remodeling the loop backbone.

Exploitation of binding energy for catalysis and design

Thyme, Summer B., et all. Nature 461, 1300-4. (2009)

The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in the center of a 20 base-pair DNA target site, with the N-terminal domain of the enzyme making extensive binding interactions with the left (-) side of the target site and the similarly structured C-terminal domain interacting with the right (+) side. Despite the approximate two-fold symmetry of the enzyme-DNA complex, we find that there is almost complete segregation of interactions responsible for substrate binding to the (-) side of the interface and interactions responsible for transition state stabilization to the (+) side.

De novo computational design of retro-aldol enzymes

Jiang, Lin, Althoff Eric A. et al, Science, 319, 1387-91. (2008)

Using new algorithms that employ hashing techniques to construct active sites for multi-step reactions, we designed retro-aldolases that employ four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a non-natural substrate. Designs that utilized an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged sidechain networks. The atomic accuracy of the design process was confirmed by the X-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model.

High Resolution Protein Structure Refinement

Qian B, Raman S, et al. Nature 450, 259-64. (2007)

A longstanding problem in computational biology is the refinement of low resolution protein structure models to more atomic-level accurate structures. A related challenge is refining low-resolution NMR models to the quality of high-resolution structures. NMR is a valuable tool for determining protein structures, particularly because it does not require crystals. But some NMR structures, especially those determined from insufficient restraints or misinterpreted data, can be incorrect. Also, the core of an NMR structure can tend to be under-packed, possibly due to overlapping spectra. To tackle both challenges, comparative model refinement and NMR structure refinement, we have been developing the Rosetta high-resolution refinement protocol. This protocol involves focusing sampling on regions of the structure that are most likely to contain errors while allowing the whole structure to relax in a physically realistic all-atom forcefield.

Computational Design of Protein-DNA Cleavage Specificity in a Homing Endonuclease

Ashworth J, Havranek JJ, et al. Nature 441, 656-9. (2006)

  High-resolution modeling of protein-DNA interactions has granted us the ability to estimate the specificities of real and hypothetical interfaces. This approach may be useful to design novel sequence-specific endonucleases for biotechnology and medicine.

The figure at left depicts the (crystallographically and biochemically validated) model of a strong switch in the basepair specificity of the endonuclease I-MsoI at a single symmetric position in the recognition region. Generalization of this method may allow us to broadly engineer novel DNA cleavage specificities using computational design.

Design of a novel globular protein fold with atomic level accuracy

Kuhlman B, Dantas G, et al. Science 302, 1364-8. (2003)

A major challenge of computational protein design is the creation of novel proteins with arbitrarily chosen three-dimensional structures. Here, we used a general computional strategy that iterates between sequence design and structure prediction to design a 93-residue alpha/beta protein called Top7 with a novel sequence and topology. Top7 was found experimentally to be folded and extremely stable, and the x-ray crystal structure of Top7 is similar (root mean square deviation equals 1.2 angstroms) to the design model. The ability to design a new protein fold makes possible the exploration of the large regions of the protein universe not yet observed in nature. Superimposition of the Top7 computational model and x-ray structure shows the remarkable atomic-level accuracy of the design (1.2 angstrom RMSD). The backbones are respresented as ribbons (computational model : helices - dark blue, strands - red; x-ray structure : helices - light blue, strands - yellow), and selected amino-acid sidechains in the protein core are represented as sticks.

Blind ab initio structure prediction of CASP3 targets

Simons, K.T., Bonneau, et al.  Proteins 3, 171-176 (1999)

To generate structures consistent with both the local and non-local interactions responsible for protein stability, 3 and 9 residue fragments of known structures with local sequences similar to the target sequence were assembled into complete tertiary structures using a Monte Carlo simulated annealing procedure (Simons, K.T. et al., J. Mol. Biol., 268:209-25, 1997 [Full Text PDF]).

Experiment and theory highlight role of native state topology in SH3 folding

Riddle, D.S., Grantcharova, V.P., et al. Nat Struct Biol 6, 1016-1204. (1999)

We use a combination of experiments, computer simulations and simple model calculations to characterize, first, the folding transition state ensemble of the src SH3 domain, and second, the features of the protein that determine its folding mechanism. Kinetic analysis of mutations at 52 of the 57 residues in the src SH3 domain revealed that the transition state ensemble is even more polarized than suspected earlier: no single alanine substitution in the N-terminal 15 residues or the C-terminal 9 residues has more than a two-fold effect on the folding rate, while such substitutions at 15 sites in the central three-stranded beta-sheet cause significant decreases in the folding rate. Molecular dynamics (MD) unfolding simulations and ab initio folding simulations on the src SH3 domain exhibit a hierarchy of folding similar to that observed in the experiments. The similarity in folding mechanism of different SH3 domains and the similar hierarchy of structure formation observed in the experiments and the simulations can be largely accounted for by a simple native state topology-based model of protein folding energy landscapes.

A correlation between folding rate and contact order

Plaxco, K. W., Simons, K. T. et al. J. Mol. Biol. 277, 985-994. (1998)

Our studies have revealed a significant correlation between the average sequence seqaration between contacting residues in the native state (contact order) and the folding rate of simple, single domain proteins.


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