The genes of all organisms have been shaped by selective pressures. The relationship between gene sequence and fitness has tremendous implications for understanding both evolutionary processes and functional constraints on the encoded proteins. Here, we have exploited deep sequencing technology to experimentally determine the fitness of all possible individual point mutants under controlled conditions for a nine-amino acid region of Hsp90. Over the past five decades, limited glimpses into the relationship between gene sequence and function have sparked a long debate regarding the distribution, relative proportion, and evolutionary significance of deleterious, neutral, and advantageous mutations. Our systematic experimental measurement of fitness effects of Hsp90 mutants in yeast, evaluated in the light of existing population genetic theory, are remarkably consistent with a nearly neutral model of molecular evolution.

Machines of protein destruction-including energy-dependent proteases and disassembly chaperones of the AAA(+) ATPase family-function in all kingdoms of life to sculpt the cellular proteome, ensuring that unnecessary and dangerous proteins are eliminated and biological responses to environmental change are rapidly and properly regulated. Exciting progress has been made in understanding how AAA(+) machines recognize specific proteins as targets and then carry out ATP-dependent dismantling of the tertiary and/or quaternary structure of these molecules during the processes of protein degradation and the disassembly of macromolecular complexes.

We report the development and initial experimental validation of a computational design procedure aimed at generating enzymelike protein catalysts called ''protozymes.'' Our design approach utilizes a ''compute and build'' strategy that is based on the physical͞chemical principles governing protein stability and catalytic mechanism. By using the catalytically inert 108-residue Escherichia coli thioredoxin as a scaffold, the histidine-mediated nucleophilic hydrolysis of p-nitrophenyl acetate as a model reaction, and the ORBIT protein design software to compute sequences, an active site scan identified two promising catalytic positions and surrounding active-site mutations required for substrate binding. Experimentally, both candidate protozymes demonstrated catalytic activity significantly above background. One of the proteins, PZD2, displayed ''burst'' phase kinetics at high substrate concentrations, consistent with the formation of a stable enzyme intermediate. The kinetic parameters of PZD2 are comparable to early catalytic Abs. But, unlike catalytic Ab design, our design procedure is independent of fold, suggesting a possible mechanism for examining the relationships between protein fold and the evolvability of protein function.A prominent goal of protein design is the generation of proteins with novel functions, including the catalytic rate enhancement of chemical reactions. The ability to design an enzyme to perform a given chemical reaction has considerable practical application for industry and medicine, particularly for the synthesis of pharmaceuticals (1). Significant progress has been made at enhancing the catalytic properties of existing enzymes through directed evolution (2). In contrast, the design of proteins with novel catalytic properties has met with relatively limited success (3-5). We present a general computational approach for the design of enzyme-like proteins with novel catalytic activities.The use of transition-state analogs as haptens to elicit catalytic Abs has been the most successful technique to date for generating novel protein catalysts (6). Natural enzymes combine transition-state stabilization with precisely oriented catalytic side chains. Although a reactive hapten has been used in the generation of an Ab with a powerful nucleophile at the active site (7), current catalytic Ab technology does not efficiently select for both catalytic side chains and tight noncovalent affinity in the same molecule. The relationship between the general backbone fold of an enzyme and its catalytic properties is not well understood. This observation is particularly relevant to catalytic Abs that are currently constrained to the Ab fold and which have yet to show catalytic activity on par with natural enzymes.Rational design efforts have recently succeeded at altering the catalytic reactions of two different enzymes (8, 9). Cyclophilin, a cis-trans isomerase of X-Pro peptide bonds, was engineered into an endopeptidase by grafting a triad of catalytic residues commonly found in serine proteases at the...

The challenge of distinguishing genetic drift from selection remains a central focus of population genetics. Time-sampled data may provide a powerful tool for distinguishing these processes, and we here propose approximate Bayesian, maximum likelihood, and analytical methods for the inference of demography and selection from time course data. Utilizing these novel statistical and computational tools, we evaluate whole-genome datasets of an influenza A H1N1 strain in the presence and absence of oseltamivir (an inhibitor of neuraminidase) collected at thirteen time points. Results reveal a striking consistency amongst the three estimation procedures developed, showing strongly increased selection pressure in the presence of drug treatment. Importantly, these approaches re-identify the known oseltamivir resistance site, successfully validating the approaches used. Enticingly, a number of previously unknown variants have also been identified as being positively selected. Results are interpreted in the light of Fisher's Geometric Model, allowing for a quantification of the increased distance to optimum exerted by the presence of drug, and theoretical predictions regarding the distribution of beneficial fitness effects of contending mutations are empirically tested. Further, given the fit to expectations of the Geometric Model, results suggest the ability to predict certain aspects of viral evolution in response to changing host environments and novel selective pressures.