Protein Dynamism and Evolvability

Tokuriki, Nobuhiko; Tawfik, Dan S.

doi:10.1126/science.1169375

Cited by 835 publications

(843 citation statements)

References 47 publications

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“…We have learned that evolution is economical: related enzymes that catalyze different reactions share conserved functional elements, including catalytic or metal-binding residues, cofactors, and key residues that modulate the chemistry (Bartlett et al 2003). And today's members of functionally diverse families exhibit the catalytic promiscuity, often as a result of their mechanisms, conformational diversity, etc., that presumably fueled their divergence in the past (Babtie et al 2010;Baier & Tokuriki, 2014;Gatti-Lafranconi & Hollfelder, 2013;Tokuriki & Tawfik, 2009). Experiments lend credence to the proposal that promiscuity underlies much of enzyme innovation.…”

Section: Nature's Approach To Chemical Innovationmentioning

confidence: 67%

The nature of chemical innovation: new enzymes by evolution

Arnold

2015

Quart. Rev. Biophys.

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Abstract. I describe how we direct the evolution of non-natural enzyme activities, using chemical intuition and information on structure and mechanism to guide us to the most promising reaction/enzyme systems. With synthetic reagents to generate new reactive intermediates and just a few amino acid substitutions to tune the active site, a cytochrome P450 can catalyze a variety of carbene and nitrene transfer reactions. The cyclopropanation, N-H insertion, C-H amination, sulfimidation, and aziridination reactions now demonstrated are all well known in chemical catalysis but have no counterparts in nature. The new enzymes are fully genetically encoded, assemble and function inside of cells, and can be optimized for different substrates, activities, and selectivities. We are learning how to use nature's innovation mechanisms to marry some of the synthetic chemists' favorite transformations with the exquisite selectivity and tunability of enzymes.

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Section: Nature's Approach To Chemical Innovationmentioning

confidence: 67%

The nature of chemical innovation: new enzymes by evolution

Arnold

2015

Quart. Rev. Biophys.

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“…6 Recent developments in NMR spectroscopy have provided new insights into the relationship between structure, function, and dynamics. 7 X-ray crystallography complements these techniques by providing detailed structural data on the nature of conformational polymorphism.…”

Section: Introductionmentioning

confidence: 99%

Automated electron‐density sampling reveals widespread conformational polymorphism in proteins

et al. 2010

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Although proteins populate large structural ensembles, X-ray diffraction data are traditionally interpreted using a single model. To search for evidence of alternate conformers, we developed a program, Ringer, which systematically samples electron density around the dihedral angles of protein side chains. In a diverse set of 402 structures, Ringer identified weak, nonrandom electron-density features that suggest of the presence of hidden, lowly populated conformations for >18% of uniquely modeled residues. Although these peaks occur at electron-density levels traditionally regarded as noise, statistically significant (P < 10 25 ) enrichment of peaks at successive rotameric v angles validates the assignment of these features as unmodeled conformations. Weak electron density corresponding to alternate rotamers also was detected in an accurate electron density map free of model bias. Ringer analysis of the high-resolution structures of free and peptide-bound calmodulin identified shifts in ensembles and connected the alternate conformations to ligand recognition. These results show that the signal in high-resolution electron density maps extends below the traditional 1 r cutoff, and crystalline proteins are more polymorphic than current crystallographic models. Ringer provides an objective, systematic method to identify previously undiscovered alternate conformations that can mediate protein folding and function.

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“…The resulting ensemble of conformational substates could give rise to promiscuity in protein-protein interactions (9) and allosteric behavior (7,8,10). In addition, large-to-small mutations that generate cavities may have played an important role in the evolution of protein function (11).…”

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confidence: 99%

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants

López

Yang

Altenbach

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The studies presented here explore the relationship between protein packing and molecular flexibility using ligand-binding cavity mutants of T4 lysozyme. Although previously reported crystal structures of the mutants investigated show single conformations that are similar to the WT protein, site-directed spin labeling in solution reveals additional conformational substates in equilibrium exchange with a WT-like population. Remarkably, binding of ligands, including the general anesthetic halothane shifts the population to the WT-like state, consistent with a conformational selection model of ligand binding, but structural adaptation to the ligand is also apparent in one mutant. Distance mapping with double electron-electron resonance spectroscopy and the absence of ligand binding suggest that the new substates induced by the cavity-creating mutations represent alternate packing modes in which the protein fills or partially fills the cavity with side chains, including the spin label in one case; external ligands compete with the side chains for the cavity space, stabilizing the WT conformation. The results have implications for mechanisms of anesthesia, the response of proteins to hydrostatic pressure, and protein engineering.EPR | site-directed spin labeling | DEER | benzene | saturation recovery G lobular proteins have overall packing densities similar to those of crystalline solids (1) but nevertheless contain cavities and pockets that can range from a few to hundreds of cubic angstroms (2, 3). These native packing defects are generally destabilizing (4, 5), and improving the packing of the protein interior may be a general way to increase protein stability. Indeed, small-to-large mutations that fill native cavities can increase stability (6-8), but with a concomitant loss of function (7,8). Thus, cavities in the protein interior can play a critical role in function. One role of cavities may be to allow alternative packing arrangements of the core. The resulting ensemble of conformational substates could give rise to promiscuity in protein-protein interactions (9) and allosteric behavior (7,8,10). In addition, large-to-small mutations that generate cavities may have played an important role in the evolution of protein function (11).Considering the potentially important role of cavities in sculpting the energy landscape of proteins, the present study was undertaken to investigate, in solution, the structural and dynamical response of a protein to engineered cavities introduced by large-tosmall mutations. T4 lysozyme (T4L) was selected for study because of the large database of crystal structures of ligand-binding cavity mutations and the corresponding thermodynamic characterization from Matthews and coworkers (4,(12)(13)(14)(15). Remarkably, comparison of the WT and mutant structures showed very little difference or limited local relaxation near the cavity, although the mutations were strongly destabilizing (4, 15).In the present study, we examined the cavity-creating mutants L121A/L133A, L133G, and W138A in sol...

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Protein Dynamism and Evolvability

Cited by 835 publications

References 47 publications

The nature of chemical innovation: new enzymes by evolution

The nature of chemical innovation: new enzymes by evolution

Automated electron‐density sampling reveals widespread conformational polymorphism in proteins

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants

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