tion of neomorphic variants. The authors ran-domly mutated four unrelated genes, cloned the resulting libraries into an expression vec-tor and used them to transform E. coli (Fig. 1). A small population (n = 360 × 4 libraries) of transformants was picked and propagated in microtiter plates. The mutant genes were expressed in the presence or absence of high levels of GroEL/GroES and were tested for native activity. The clones that evinced wild-type–like activities were pooled and randomly mutated for the next round of screening. After four rounds of drift, many (~30%) of the pro-tein variants were chaperonin dependent. Not surprisingly, chaperonin dependence did not evolve in control drifts without GroEL/GroES overexpression. The GroEL/GroES-dependent evolvants collectively contained more muta-tions, particularly in the core residues, than

engineered thermostable proteins were more robust (able to tolerate mutations) and evolv-able (able to evolve new structural features and function) than their marginally stable wild-type counterparts2. Bloom et al. and Bershtein et al. separately showed that artificial genetic drift (directed evolution of populations of pro-teins under high mutation rates and selection for wild-type activity) tends to favor the selec-tion of stabilizing mutations3,4. Chaperones offer an alternative way to stabilize proteins. Previous workers have demonstrated that the chaperonin GroEL/GroES can rescue unstable mutant proteins and enable cells to withstand higher mutational loads5,6.

Tokuriki and Tawfik have now shown that overexpression of GroEL/GroES permits the folding of otherwise inviable mutant proteins in Escherichia coli, thereby accelerating the fixa-

Protein engineers redesign proteins to improve their utility as biocatalysts, biologic therapies, materials and diagnostics. They generally use rational design (structure-based site-directed mutagenesis) or directed evolution (iterated cycles of random mutagenesis and high-throughput screening) strategies to modify the structures and functions of wild-type enzymes. The latter approach has inspired the development of sophisticated techniques for constructing sequence-variant libraries and evaluating clones in high-throughput assays. In the absence of an evolutionary model, how-ever, the utility of any such technique can only be evaluated through experimental trial and error. Protein engineers can take the guess-work out of directed evolution by working within the framework of existing theory. For example, Tokuriki and Tawfik showed recently that chaperonin overexpression improves the efficiency of laboratory evolution1. Their new technique is effective, easy to implement and compatible with most existing protocols. More importantly, their results elaborate a badly needed structural model of adaptive enzyme evolution.

Charles Darwin established the paradigm of natural selection in 1859, but our under-standing of adaptation at the molecular level remains rudimentary. Protein sequences diverge over evolutionary timescales, but most mutations that survive selection do not affect fitness. Amino acid changes are more likely to be destabilizing than not, but a mutation will be deleterious to the organism only if it causes the overall stability of the tertiary structure to fall below the threshold necessary to retain activity at physiological temperatures. Most wild-type proteins are only marginally stable, so many random mutants are unable to retain their folds. Bloom et al. showed in 2006 that

How evolving enzymes can beat the heat and avoid defeatIchiro Matsumura & Andrei A Ivanov

Random mutations usually destabilize protein tertiary structure. Overexpression of the chaperonin GroEL/GroES protects evolving proteins from the destabilizing effects of adaptive mutations and improves the quantity and quality of enzyme variants with modified function.

Ichiro Matsumura and Andrei A. Ivanov are in the Department of Biochemistry, Center for Fundamental and Applied Molecular Evolution, Emory University School of Medicine, Atlanta, Georgia, USA. e-mail: imatsum@emory.edu

Randommutagenesis

Randommutagenesis

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GroEL/GroES-independentenzyme withnew function Evolution without

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GroEL/GroES

GroEL/GroES

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Figure 1 Chaperonin overexpression accelerates adaptive protein evolution. Wild-type protein-coding genes are randomly mutated and the resulting libraries expressed in E. coli. The overexpression of the chaperonin GroEL/GroES rescues some mutants, including those with new activities, that would otherwise have folded incorrectly. The enzyme variants are evaluated in high-throughput assays to assess the native activity (artificial genetic drift) or a new one (directed evolution). Duplicate assays of clones with or without overexpressed GroEL/GroES enable the identification of chaperonin-dependent variants. Afterward, variants that retain their new functions without GroEL/GroES overexpression can be evolved.

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tein. The GroEL/GroES expression vector used in this study (pGro7) is commercially available, so it can be incorporated into any directed evo-lution experiment. Indeed, protein engineers have devised many ways to improve the folding of recombinant proteins in E. coli, including decreased temperatures, the use of osmolytes7 and fusion to stable domains8. These tech-niques, perhaps in combination with GroEL/GroES overexpression, should further acceler-ate the artificial evolution of proteins with new functions.

1. Tokuriki, N. & Tawfik, D.S. Nature 459, 668–673 (2009).

2. Bloom, J.D., Labthavikul, S.T., Otey, C.R. & Arnold, F.H. Proc. Natl. Acad. Sci. USA 103, 5869–5874 (2006).

3. Bershtein, S., Goldin, K. & Tawfik, D.S. J. Mol. Biol. 379, 1029–1044 (2008).

4. Bloom, J.D. et al. BMC Biol. 5, 29 (2007).5. Rutherford, S.L. Nat. Rev. Genet. 4, 263–274

(2003).6. Tomala, K. & Korona, R. Biol. Direct 3, 5 (2008).7. Ignatova, Z. & Gierasch, L.M. Proc. Natl. Acad. Sci. USA

103, 13357–13361 (2006).8. Panavas, T., Sanders, C. & Butt, T.R. Methods Mol. Biol.

497, 303–317 (2009).

an evolving protein. The constitutive overex-pression of this chaperonin would impose no fitness cost upon E. coli5, but it would perma-nently lower the thermodynamic threshold for protein unfolding and permit the evolution of even less stable proteins. A population would benefit more from transiently increasing the evolvability of its proteins by overexpressing GroEL/GroES during times of stress; normal GroEL/GroES expression thereafter could favor the evolution of chaperonin-independent forms that retain newly evolved functions.

Protein engineers use directed evolution to create better biocatalysts for the synthe-sis of unnatural (and chiral) compounds. Unfortunately, a proliferation of techniques has fostered confusion and a trial-and-error approach to experimental design. The key to evaluating techniques, and to interpreting data, is the development of a structural model of protein adaptation. Tokuriki and Tawfik have articulated such a model and demonstrated an easy way to regulate the evolvability of any pro-

their chaperonin-independent sibs. Tokuriki also evolved a larger population (n = 10,000) of phosphotriesterase variants either with or without GroEL/GroES overexpression. The excess chaperonin actuated the evolution of a larger number of variants that reacted with a new substrate, 6-naphthylhexanoate. Some showed ten times the activity and specificity for the new substrate of the best clones that evolved in the absence of GroEL/GroES over-expression.

These results are consistent the hypoth-esis that chaperones regulate evolvability. The chaperone Hsp90 stabilizes signal transduc-tion proteins, so the reduction of Hsp90 activ-ity in a population of Drosophila melanogaster unveils previously silent phenotypic variation. The fine-tuning of Hsp90 activity, perhaps by changing the mix of substrates competing for finite numbers of Hsp90 active sites, would gen-erate variation only in the most atypical indi-viduals within the population5. GroEL/GroES can similarly smooth the fitness landscape of

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