Directed evolution converts subtilisin E into a functional equivalent of thermitase

Zhao, Huimin; Arnold, Frances H.

doi:10.1093/protein/12.1.47

Cited by 308 publications

(218 citation statements)

References 37 publications

Supporting

Mentioning

197

Contrasting

Unclassified

Order By: Relevance

“…Among the subset of sequences that do stably fold, the simple statistical reality that marginally stable sequences are far more abundant than highly stable sequences causes evolution to further confine itself mostly to sequences with stabilities far less than that of the most stable sequence (Arnold et al 2001;Taverna and Goldstein 2002a;this work). This fact is amply demonstrated by engineering experiments that have greatly increased the stability of natural proteins without sacrificing any of their functional properties (Serrano et al 1993;Giver et al 1998;van den Burg et al 1998;Zhao and Arnold 1999). Therefore, although the distribution of DDG values certainly varies widely among all sequences, it is still reasonable to assume that it is relatively constant among those sequences visited by natural evolution.…”

Section: Resultsmentioning

confidence: 99%

Thermodynamics of Neutral Protein Evolution

2007

View full text Add to dashboard Cite

Naturally evolving proteins gradually accumulate mutations while continuing to fold to stable structures. This process of neutral evolution is an important mode of genetic change and forms the basis for the molecular clock. We present a mathematical theory that predicts the number of accumulated mutations, the index of dispersion, and the distribution of stabilities in an evolving protein population from knowledge of the stability effects (DDG values) for single mutations. Our theory quantitatively describes how neutral evolution leads to marginally stable proteins and provides formulas for calculating how fluctuations in stability can overdisperse the molecular clock. It also shows that the structural influences on the rate of sequence evolution observed in earlier simulations can be calculated using just the singlemutation DDG values. We consider both the case when the product of the population size and mutation rate is small and the case when this product is large, and show that in the latter case the proteins evolve excess mutational robustness that is manifested by extra stability and an increase in the rate of sequence evolution. All our theoretical predictions are confirmed by simulations with lattice proteins. Our work provides a mathematical foundation for understanding how protein biophysics shapes the process of evolution.

show abstract

Section: Resultsmentioning

confidence: 99%

Thermodynamics of Neutral Protein Evolution

2007

View full text Add to dashboard Cite

show abstract

“…Several thermodynamically stable proteins have been documented to achieve thermostability step-wise, with individual amino acid changes providing incremental, additive contributions (Serrano et al 1993;Zhang et al 1995;Zhao and Arnold 1999). Our RNA achieves thermostability in a very different way: Contributions by individual mutations are context dependent, nonadditive, but highly cooperative.…”

Section: Strategies Used By Proteins and Rnas To Achieve Thermostabilitymentioning

confidence: 91%

“…Many thermostable mutations in proteins are located on the surface (Zhao and Arnold 1999;Yano and Poulos 2003). They either introduce new electrostatic interactions or optimize existing ones (Chakravarty and Varadarajan 2002).…”

Section: Strategies Used By Proteins and Rnas To Achieve Thermostabilitymentioning

confidence: 99%

Comparison of crystal structure interactions and thermodynamics for stabilizing mutations in the Tetrahymena ribozyme

Guo¹,

Gooding²,

Cech³

2006

RNA

View full text Add to dashboard Cite

Although general mechanisms of RNA folding and catalysis have been elucidated, little is known about how ribozymes achieve structural stability at high temperature. A previous in vitro evolution experiment identified a small number of mutations that significantly increase the thermostability of the tertiary structure of the Tetrahymena ribozyme. Because we also determined the crystal structure of this thermostable ribozyme, we have for the first time the opportunity to compare the structural interactions and thermodynamic contributions of individual nucleotides in a ribozyme. We investigated the contribution of five mutations to thermostability by using temperature gradient gel electrophoresis. Unlike the case with several well-studied proteins, the effects of individual mutations on thermostability of this RNA were highly context dependent. The three most important mutations for thermostability were actually destabilizing in the wild-type background. A269G and A304G contributed to stability only when present as a pair, consistent with their proximity in the ribozyme structure. In an evolutionary context, this work supports and extends the idea that one advantage of protein enzyme systems over an RNA world is the ability of proteins to accumulate stabilizing single-site mutations, whereas RNA may often require much rarer double mutations to improve the stability of both its tertiary and secondary structures.

show abstract

“…Thermostability, for example, could be conferred by any one of the at least three different genetic elements. Because of the importance of proteolytic enzymes, directed evolution of proteases and peptidases remains one of the most actively pursued research areas (10,12,34,100,160,210,211,285,297,304,(327)(328)(329)349).…”

Section: Directed Evolution Of Biochemical Catalystsmentioning

confidence: 99%

Laboratory-Directed Protein Evolution

Yuan

Kurek

English

et al. 2005

Microbiol Mol Biol Rev

176

View full text Add to dashboard Cite

Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to “evolve” in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences

show abstract

Directed evolution converts subtilisin E into a functional equivalent of thermitase

Cited by 308 publications

References 37 publications

Thermodynamics of Neutral Protein Evolution

Thermodynamics of Neutral Protein Evolution

Comparison of crystal structure interactions and thermodynamics for stabilizing mutations in the Tetrahymena ribozyme

Laboratory-Directed Protein Evolution

Contact Info

Product

Resources

About