Research
Neutral drifts - promoters of protein evolvability
Most of the genetic diversity of this planet is the outcome of neutral, non-adaptive drifts. Despite the ubiquity of such neutral changes, we know little about them, and in particular, we know little about whether, and how, neutral changes can promote adaptive evolution, namely the evolution of new protein functions.
A neutral drift we performed with the enzyme serum paraoxonase (PON1), demonstrated that the potential for adaptation develops dramatically when the neutral network of a protein expands. Almost half of the neutral PON1 variants characterized exhibited significant changes in promiscuous activities, and PON1 neutral variants with one, or even two, mutations that mediate a new phenotype (aryl esterase, thiolactonase, phosphotriesterase, or "drug resistance") were readily identified (for a perspective article see DePristo, HFSP (2007).)
Neutral sequence changes in PON1’s tertiary structures, and putative neutral network: An accelerated neutral drift performed with a lactonase dubbed PON1 was found to introduce a wide range of apparently neutral mutations in the periphery, and within the active site, and thus created an expanded neutral network (grey circle) from which various new specificities can stem (designated by peripheral crosses in various colours) (Amitai HFSP, (2007)).
Other neutral drifts we performed involved TEM-1 beta-lactamase. We measured how the fitness (activity, stability, etc.) of TEM-1 changes as mutations accumulate. Sequence analysis of the drifting libraries indicated that mutations that act as 'global suppressors' were enriched throughout the neutral drift. These mutations increased TEM-1's stability, suppressed the effect of a broad range of destabilizing mutations, and thereby significantly increased TEM-1 evolvability. Interestingly, all identified global suppressors emerged in positions where the sequence of TEM-1 deviates from its family consensus, and/or predicted ancestor, and comprise back-to-consensus/ancestor mutations. We also showed that TEM-1 rate of adaptation (the frequency of variants exhibiting a new antibiotic specificity) is maximal following a neutral drift, namely, when selection for the original function was not completely removed, but partially relieved, thus enabling the accumulation of potentially adaptive mutations while purging a large fraction of otherwise deleterious mutations.
We preformed neutral drifts of several other enzymes in the absence and in presence of chaperone over-expression, to examine the effect of chaperone buffering on the number and type of mutations.
The pleiotropic effects of mutations revealed by these experiments are also affecting our understanding iof the mechanisms by which new proteins and genes diverge from existing ones.
We have also demonstrated the utility of neutral drifts for directed evolution. Small libraries (<1,000 variants) can be obtained by neutral drifts that maintain the protein's original function, yielding highly polymorphic, stable and evolvable variants that can be subsequently screened for new enzymatic functions. To this end, the drifted enzyme was fused to an optimized GFP that reported levels of soluble, functional enzyme, thus enabling selection by flow cytometry and identification of enzyme variants exhibiting improved specific and total activities.
Selected publications
Bershtein S, Segal M, Bekerman R, Tokuriki N, Tawfik DS. "Robustness-epistasis link shapes the fitness landscape of a randomly drifting protein." Nature (2006) Dec 14;444(7121):929-932.
Amitai G, Devi Gupta R, Tawfik DS. "Latent evolutionary potentials under the neutral mutational drift of an enzyme." HFSP Journal, May (2007);1(1):67-78.
Bershtein S, Tawfik DS. “Ohno's model revisited: measuring the frequency of potentially adaptive mutations under various mutational drifts.” Mol Biol Evol. (2008) Nov;25(11):2311-2318.
Shimon Bershtein, Korina Goldin, Tawfik DS. "Intense Neutral Drifts Yield Robust and Evolvable Consensus Proteins." J Mol Biol. (2008) June;379(5):1029–1044.
Gupta RD, Tawfik DS. “Directed enzyme evolution via small and effective neutral drift libraries.” Nat Methods. (2008) Nov;5(11):939-942.
Tokuriki N, Tawfik DS. “Protein dynamism and evolvability.” Science (2009) Apr 10;324(5924):203-207.
Soskine M, Tawfik DS. “Mutational effects and the evolution of new protein functions.” Nat Rev Genet. (2010) Aug;11(8):572-582.
Tóth-Petróczy Á, Tawfik DS. “Slow protein evolutionary rates are dictated by surface-core association.” PNAS (2011) June; 108 (27) 11151-11156.
Alcolombri U, Elias M, Tawfik DS. “Directed Evolution of Sulfotransferases and Paraoxonases by Ancestral Libraries.” JMB (2011) Aug 26;411(4):837-53..
Salverda ML, Dellus E, Gorter FA, Debets AJ, van der Oost J, Hoekstra RF, Tawfik DS, de Visser JA. “Initial mutations direct alternative pathways of protein evolution.” PLoS Genet (2011) Mar;7(3):e1001321.
