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Proteins are only moderately stable. It has long been debated whether this narrow range of stabilities is solely a result of neutral drift towards lower stability or purifying selection against excess stability is also at work - for which no experimental evidence was found so far. Here we show that mutations outside the active site in the essential E. coli enzyme adenylate kinase result in stability-dependent increase in substrate inhibition by AMP, thereby impairing overall enzyme activity at high stability. Such inhibition caused substantial fitness defects not only in the presence of excess substrate but also under physiological conditions. In the latter case, substrate inhibition caused differential accumulation of AMP in the stationary phase for the inhibition prone mutants. Further, we show that changes in flux through Adk could accurately describe the variation in fitness effects. Taken together, these data suggest that selection against substrate inhibition and hence excess stability may have resulted in a narrow range of optimal stability observed for modern proteins., Comment: 30 pages, 6 figures, 1 table, Supplementary figures and tables - 6 pages

Mutations in a microbial population can increase the frequency of a genotype not only by increasing its exponential growth rate, but also by decreasing its lag time or adjusting the yield (resource efficiency). The contribution of multiple life-history traits to selection is a critical question for evolutionary biology as we seek to predict the evolutionary fates of mutations. Here we use a model of microbial growth to show there are two distinct components of selection corresponding to the growth and lag phases, while the yield modulates their relative importance. The model predicts rich population dynamics when there are tradeoffs between phases: multiple strains can coexist or exhibit bistability due to frequency-dependent selection, and strains can engage in rock-paper-scissors interactions due to non-transitive selection. We characterize the environmental conditions and patterns of traits necessary to realize these phenomena, which we show to be readily accessible to experiments. Our results provide a theoretical framework for analyzing high-throughput measurements of microbial growth traits, especially interpreting the pleiotropy and correlations between traits across mutants. This work also highlights the need for more comprehensive measurements of selection in simple microbial systems, where the concept of an ordinary fitness landscape breaks down.

Considerable mechanistic insight has been gained into amyloid aggregation; however, a large class of non-amyloid protein aggregates are considered 'amorphous,' and in most cases little is known about their mechanisms. Amorphous aggregation of {\gamma}-crystallins in the eye lens causes a widespread disease of aging, cataract. We combined simulations and experiments to study the mechanism of aggregation of two {\gamma}D-crystallin mutants, W42R and W42Q - the former a congenital cataract mutation, and the latter a mimic of age-related oxidative damage. We found that formation of an internal disulfide was necessary and sufficient for aggregation under physiological conditions. Two-chain all-atom simulations predicted that one non-native disulfide in particular, between Cys32 and Cys41, was likely to stabilize an unfolding intermediate prone to intermolecular interactions. Mass spectrometry and mutagenesis experiments confirmed the presence of this bond in the aggregates and its necessity for oxidative aggregation under physiological conditions in vitro. Mining the simulation data linked formation of this disulfide to extrusion of the N-terminal \b{eta}-hairpin and rearrangement of the native \b{eta}-sheet topology. Specific binding between the extruded hairpin and a distal \b{eta}-sheet, in an intermolecular chain reaction similar to domain swapping, is the most probable mechanism of aggregate propagation., Comment: *equal contribution; {\dag}corresponding authors

The catalytic conversion of ATP and AMP to ADP by adenylate kinase (ADK) involves large amplitude, ligand induced domain motions, involving the opening and the closing of LID and NMP domains, during the repeated catalytic cycle. We discover and analyze an interesting dynamical coupling between the motions of the two domains during the opening, using large scale atomistic molecular dynamics trajectory analysis, covariance analysis and multi-dimensional free energy calculations with explicit water. Initially, the LID domain must open by a certain amount before the NMP domain can begin to open. Dynamical correlation map shows interesting cross-peak between LID and NMP domain which suggests the presence of correlated motion between them. This is also reflected in our calculated two dimensional free energy surface contour diagram which has an interesting elliptic shape, revealing a strong correlation between the opening of the LID domain and that of the NMP domain. Our free energy surface of the LID domain motion is rugged due to interaction with water and the signature of ruggedness is evident in the observed RMSD variation and its fluctuation time correlation functions. We develop a correlated dynamical disorder type theoretical model to explain the observed dynamic coupling between the motions of the two domains in ADK. Our model correctly reproduces several features of the cross-correlation observed in simulations., Comment: 34 pages, 9 figures

The catalytic conversion ATP + AMP -> 2ADP by the enzyme adenylate kinase (ADK) involves the binding of one ATP molecule to the LID domain and one AMP molecule to the NMP domain. The latter is followed by a phosphate transfer, and then the release of two ADP molecules. We have computed a novel two dimensional configurational free energy surface (2DCFES), with one reaction coordinate each for the LID and the NMP domain motions, with explicit interactions with water. Our computed 2DCFES clearly reveals the existence of a stable half-open-half-closed (HOHC) intermediate state of the enzyme. Cycling of the enzyme through the HOHC state reduces the conformational free energy barrier for the reaction by about 20 kJ/mol. We find that the stability of the half-open-half-closed state (missed in all earlier studies with implicit solvent model) is largely because of the increase of specific interactions of the polar amino acid side chains with water, particularly with the arginine and the histidine residues. Free energy surface of the LID domain is rather rugged, which can conveniently slow down LID's conformational motion, thus facilitating a new substrate capture after the product release in the catalytic cycle., Comment: 28 pages, 10 figures