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Protein Evolution: Innovative Chaps chaperonin levels than at normal levels of expression. Many of the chaperonin-compensated proteins Mutations in proteins allow functional innovation, but can be critically had folding defects appearing as destabilizing. Recent work shows how chaperonins can rescue innovative decreased solubility in the absence mutants, with implications for protein engineering and adaptive evolution. of GroEL/S. These findings suggest that the chaperonins have the D. Allan Drummond were measured independently for potential to promote accumulation a large number of enzymes bearing of genetic diversity. Like hikers on a cliff edge, proteins tend random mutations, almost all folded How GroEL/S rescues mutant to be one step away from a disastrous proteins at least retained parental proteins with folding defects has been fall. Over evolutionary time, proteins function [2]. Amino acid changes that demonstrated in a series of studies by hike in sequence space [1], with each preserve stable folding represent Teschke, King and colleagues [8–11]. step corresponding to a sequence opportunities for functional innovation, Using a set of temperature-sensitive change, most often a single amino-acid and indeed, in the same study, a folding (tsf) mutants of the substitution. Each step offers the more-stable enzyme variant acquired bacteriophage P22 coat protein, a opportunity, however unlikely, to novel functions at a far higher rate model substrate which is amenable to acquire improved or novel activity, yet than its less-stable counterpart [2]. detailed folding studies and has a clear also carries the risk, often quite likely, Conversely, when directed evolution of functional assay, they have shown that of becoming unstable (Figure 1A), a fatty-acid hydroxylase toward activity tsf substitutions that are intolerable in leading to misfolding and degradation. on short-chain alkanes stalled, it was the absence of GroEL/S chaperonins For protein engineers looking to because the innovative mutations had can be rescued in the presence of the explore new functions, and perhaps destabilized the enzyme such that few, chaperonins [8]. Such rescue occurs for organisms faced with a new if any, additional mutations could be through direct interaction between the environmental challenge, the most tolerated [5]. Stabilizing the enzyme, chaperonins and the destabilized useful substitutions may at the same however, enabled the mutational protein, preventing aggregation time be the most disruptive to protein march toward new substrates to monitored by decreased solubility [9]. stability [2,3]. Innovation, in short, is resume [5]. Another way to accelerate Moreover, suppressor mutations costly. Recent work [4] suggests that acquisition of new protein functions in tsf mutants facilitate the this cost of innovation can be mitigated would be to provide a system to buffer chaperonin2substrate interaction by helper proteins called chaperonins the effects of destabilizing mutations [10], and chaperonins act to bias which help some of their protein clients [2], and chaperones offer a perfect polypeptide flux away from to accumulate more, and sometimes example of such a system. aggregation of a folding intermediate more useful, substitutions than they The bacterial chaperonins GroEL and toward folding and assembly could in the absence of folding and GroES form a cavity in which [10,11]. Interestingly, wild-type P22 assistance. amino-acid chains can attempt to coat protein is not a GroEL/S client, fold while protected from the crowded whereas its tsf mutants are [8,9]. Stability to Folding to Activity intracellular milieu. About 10% of Most proteins fold with a net stability soluble Escherichia coli proteins are Rescuing Innovative Mutants equivalent to that of a handful of clients of GroEL/S [6], which are known Although the P22 coat-protein work hydrogen bonds or, importantly, to the to suppress a wide range of mutations anticipates many more recent results, effect of a single destabilizing amino [7]. By performing mutation- this system lacks a screen for acid substitution. Critically destabilized accumulation experiments on four functional diversification, leaving the proteins cannot maintain the folded soluble enzymes under conditions possible functional effects of the state that confers their biological where GroEL/S were alternately additional mutations rescued by activity, and tend to aggregate, often overexpressed or expressed normally, GroEL/S unaddressed. By contrast, observed macroscopically as a sharp Tokuriki and Tawfik [4] demonstrated enzymes have obvious diversification decrease in solubility. In a rare study that a larger fraction of mutant proteins potential due to wide-ranging activities where protein folding and function retained activity in the presence of high on different substrates. Focusing Dispatch R741

absence of chaperonin The optimal method for isolating Novel Wild-type Novel overexpression. These results identify improved proteins — both during activity activity activity chaperonins as a buffer for protein evolution and during directed destabilizing yet functionally innovative protein design — remains an active area A mutations [2], and reflect the simple of study. Chaperonin overexpression idea that the major predictor of [4] and pre-stabilization of proteins [2] improved function in a large collection provide alternative, and compatible, of mutant proteins is simply the strategies for improving the yield of number of folded polypeptides novel enzymes (Figure 1). Notably, with unique sequences in that Tokuriki and Tawfik [4] report that collection [12]. in vitro evolution of the model proteins B The notion that chaperones could (and known GroEL/S clients) GFP and buffer evolutionarily important genetic b-lactamase did not support the trends variation, conferring ‘phenotypic they observed for the four enzymes capacitance’ in the sense of producing they discuss in detail. How chaperonin a consistent phenotypic output given activity influences organismal evolution variable genetic inputs, was introduced remains even more murky. But the by Rutherford and Lindquist [13] in a chaperonin-facilitated molecular study of the effects of the chaperone innovation detailed in recent studies C Hsp90 on Drosophila melanogaster provides tantalizing fodder for an morphology. In those experiments, experimental demonstration of

GroEL/S startling lineage-specific phenotypic capacitance — the morphological variation arose upon emergence of chaperone-buffered inhibition of Hsp90, whose clients phenotypic novelty — in laboratory include many transcription factors. evolution. Current Biology Similar results were obtained in Arabidopsis thaliana [14], where alleles References Figure 1. Helping to stabilize innovative 1. Maynard Smith, J. (1970). Natural selection with Hsp90-dependent phenotypes and the concept of a protein space. Nature 225, mutations. are common [15]. The evolutionary 563–564. (A) Wild-type proteins (blue) are typically argument holds that the genotypic 2. Bloom, J.D., Labthavikul, S., Otey, C.R., and Arnold, F.H. (2006). Protein stability promotes marginally stable, and mutants (red), which and resulting phenotypic variation have a chance to access novel activities, evolvability. Proc. Natl. Acad. Sci. USA 103, suppressed by the chaperone could 5869–5874. tend to be destabilized, often leading to 3. Tokuriki, N., Stricher, F., Serrano, L., and loss of fold and function (right). (B) Pre-stabi- be adaptive in some environments. Tawfik, D.S. (2008). How protein stability and lization of a wild-type enzyme makes more The chaperone’s activity would thus new functions trade off. PLoS Comput. Biol. destabilizing mutations tolerable, allowing allow a population to maintain a 4, e1000002. 4. Tokuriki, N., and Tawfik, D.S. (2009). access to novel activities. (C) Chaperonins hidden portfolio of ‘genetic wagers’ Chaperonin overexpression promotes genetic help destabilized mutants to fold, allowing which could enable faster adaptation variation and enzyme evolution. Nature 459, access to novel activities. 668–673. upon environmental change — so long 5. Fasan, R., Meharenna, Y.T., Snow, C.D., as such changes transiently suppress Poulos, T.L., and Arnold, F.H. (2008). on a variant of the enzyme the chaperone’s activity, revealing Evolutionary history of a specialized p450 propane monooxygenase. J. Mol. Biol. 383, phosphotriesterase from cryptic variation and, in a sense, 1069–1080. Pseudomonas sp., Tokuriki and placing previously hidden ‘wagers’ 6. Kerner, M.J., Naylor, D.J., Ishihama, Y., Maier, T., Chang, H.-C., Stines, A.P., Tawfik [4] used two rounds of on the table. Georgopoulos, C., Frishman, D., mutagenesis and screening in order The new GroEL/S overexpression Hayer-Hartl, M., Mann, M., et al. (2005). to select phosphotriesterase mutants study provides a molecular counterpart Proteome-wide analysis of chaperonin-dependent protein folding with improved hydrolysis activity to the idea of morphological ‘hopeful in Escherichia coli. Cell 122, 209–220. on the chromogenic ester monsters’ — a set of mutant proteins 7. van Dyk, T.K., Gatenby, A.A., and LaRossa, R.A. (1989). Demonstration by 2-naphthylhexanoate (2-NPH), a bearing novel mutations and, in at least genetic suppression of interaction of GroE poor substrate for the starting enzyme. one case, enhanced enzymatic products with many proteins. Nature 342, Without GroEL/S overexpression, activities (hope) along with structural 451–453. 8. Gordon, C.L., Sather, S.K., Casjens, S., and the mutant libraries yielded an up to defects (monstrosity) which require King, J. (1994). Selective in vivo rescue by 3.4-fold improvement in enzymatic chaperonin assistance. Hsp90 workers GroEL/ES of thermolabile folding intermediates to phage P22 structural proteins. J. Biol. Chem. activity with up to a 4.2-fold higher tend to assert that the stress of a 269, 27941–27951. substrate selectivity. With GroEL/S changing environment will overload 9. Nakonechny, W.S., and Teschke, C.M. (1998). overexpression, however, mutants with or otherwise reduce the efficacy of GroEL and GroES control of substrate flux in the in vivo folding pathway of phage an up to 44-fold higher esterase activity the chaperone [15], revealing adaptive P22 coat protein. J. Biol. Chem. 273, and a 54-fold higher selectivity were variation, whereas Tokuriki and 27236–27244. 10. Parent, K.N., Ranaghan, M.J., and isolated. Importantly, a first-round Tawfik [4] note that stress induces Teschke, C.M. (2004). A second-site mutation accumulated in the presence chaperone expression, allowing suppressor of a folding defect functions via of GroEL/S overexpression conferred mutant proteins to survive and interactions with a chaperone network to improve folding and assembly in vivo. Mol. a nearly 12-fold improvement in contribute adaptively with higher Microbiol. 54, 1036–1050. esterase activity but also compromised probability [4]. Future work must 11. Doyle, S.M., Anderson, E., Zhu, D., Braswell, E.H., and Teschke, C.M. (2003). folding, such that the amount of soluble reconcile these plausible but Rapid unfolding of a domain populates an protein decreased by 2.4-fold in the opposing hypotheses. aggregation-prone intermediate that can be Current Biology Vol 19 No 17 R742

recognized by GroEL. J. Mol. Biol. 332, 14. Queitsch, C., Sangster, T.A., and Lindquist, S. FAS Center for Systems Biology Northwest 937–951. (2002). Hsp90 as a capacitor of phenotypic Building, Rm. 431, 52 Oxford St, Harvard 12. Drummond, D.A., Iverson, B.L., Georgiou, G., and variation. Nature 417, 618–624. University, Cambridge, MA 02138, USA. Arnold, F.H. (2005). Why high-error-rate random 15. Sangster, T.A., Salathia, N., Undurraga, S., mutagenesis libraries are enriched in functional Milo, R., Schellenberg, K., Lindquist, S., and E-mail: [email protected] and improvedproteins. J. Mol. Biol. 350, 806–816. Queitsch, C. (2008). HSP90 affects the 13. Rutherford, S.L., and Lindquist, S. (1998). expression of genetic variation and Hsp90 as a capacitor for morphological developmental stability in quantitative traits. evolution. Nature 396, 336–342. Proc. Natl. Acad. Sci. USA 105, 2963–2968. DOI: 10.1016/j.cub.2009.07.039