Taisong Zou

Evolution of Conformational Dynamics Determines the Conversion of a Promiscuous Generalist into a Specialist Enzyme

β-Lactamases are produced by many modern bacteria as a mechanism of resistance toward β-lactam antibiotics, the most common antibiotics in use. β-Lactamases, however, are ancient enzymes that originated billions of years ago. Recently, proteins corresponding to 2- to 3-Gy-old Precambrian nodes in the evolution of Class A β-lactamases have been prepared and shown to be moderately efficient promiscuous catalysts, able to degrade a variety of antibiotics with catalytic efficiency levels similar to those of an average modern enzyme. Remarkably, there are few structural differences (in particular at the active-site regions) between the resurrected enzymes and a penicillin-specialist modern β-lactamase. Here, we propose that the ancestral promiscuity originates from conformational dynamics. We investigate the differences in conformational dynamics of the ancient and extant β-lactamases through MD simulations and quantify the contribution of each position to functionally related dynamics through Dynamic Flexibility Index. The modern TEM-1 lactamase shows a comparatively rigid active-site region, likely reflecting adaptation for efficient degradation of a specific substrate (penicillin), whereas enhanced deformability at the active-site neighborhood in the ancestral resurrected proteins likely accounts for the binding and subsequent degradation of antibiotic molecules of different size and shape. Clustering of the conformational dynamics on the basis of Principal Component Analysis is in agreement with the functional divergence, as the ancient β-lactamases cluster together, separated from their modern descendant. Finally, our analysis leads to testable predictions, as sites of potential relevance for the evolution of dynamics are identified and mutations at those sites are expected to alter substrate-specificity.

A hinge migration mechanism unlocks the evolution of green-to-red photoconversion in GFP-like proteins.

In proteins, functional divergence involves mutations that modify structure and dynamics. Here we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. Crystallographic structures were determined for several reconstructed ancestral proteins belonging to a GFP class frequently employed in superresolution microscopy. Their chain flexibility was analyzed using molecular dynamics and perturbation response scanning. The green-to-red photoconvertible phenotype appears to have arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the β barrel fold. The allosterically coupled mutational sites provide active site conformational mobility via epistasis. We propose that light-induced chromophore twisting is enhanced in a reverse-protonated subpopulation, activating internal acid-base chemistry and backbone cleavage to enlarge the chromophore. Dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities.

Proteome Folding Kinetics Is Limited by Protein Halflife

How heterogeneous are proteome folding timescales and what physical principles, if any, dictate its limits? We answer this by predicting copy number weighted folding speed distribution – using the native topology – for E.coli and Yeast proteome. E.coli and Yeast proteomes yield very similar distributions with average folding times of 100 milliseconds and 170 milliseconds, respectively. The topology-based folding time distribution is well described by a diffusion-drift mutation model on a flat-fitness landscape in free energy barrier between two boundaries: i) the lowest barrier height determined by the upper limit of folding speed and ii) the highest barrier height governed by the lower speed limit of folding. While the fastest time scale of the distribution is near the experimentally measured speed limit of 1 microsecond (typical of barrier-less folders), we find the slowest folding time to be around seconds (8 seconds for Yeast distribution), approximately an order of magnitude less than the fastest halflife (approximately 2 minutes) in the Yeast proteome. This separation of timescale implies even the fastest degrading protein will have moderately high (96%) probability of folding before degradation. The overall agreement with the flat-fitness landscape model further hints that proteome folding times did not undergo additional major selection pressures – to make proteins fold faster – other than the primary requirement to “sufficiently beat the clock” against its lifetime. Direct comparison between the predicted folding time and experimentally measured halflife further shows 99% of the proteome have a folding time less than their corresponding lifetime. These two findings together suggest that proteome folding kinetics may be bounded by protein halflife.

174 Mechanism of protein evolution: conformational dynamics and allostery

of observed structural patterns as modules or “building blocks” in large proteins (higher secondary structure content). From structural descriptor analysis, observed patterns are found to be within similar deviation, however frequent patterns are found to be distinctly occurring in diverse functions e.g. in enzymatic classes and reactions (Khan & Ghosh, in press). We have further analyzed and compared the kinetic accessibility of structural patterns using structure based models (SBM) and Go-like potential (Lammert, Schug, & Onuchic, 2009). Monitoring the folding/unfolding transitions, our result shows that homogeneity in tertiary contacts contributes to the distinct transition phases (unfolding/folding basins). The balance between tertiary and local contacts contributes to the cooperative nature of folding, which are found to be prominent characteristic in prevalent structural patterns.

Effect of Ribosomal Surface on Nascent Chain Dynamics

Protein synthesis inside the cell is remarkably complex. Various factors like chaperones, enzymatic processing and ribosome can profoundly affect the sequence and nature of the events leading to protein folding in the natural environment. Recent experiments confirm this fact and indicate that there is continuous cross-talk between the ribosome and the translated nascent chain as it emerges out of the exit tunnel. However, the specific ribosomal features that regulate this process at the molecular level are still unclear. Using molecular dynamics simulations, we study the effect of the ribosomal surface by comparing the folding behavior of ribosome-bound with that of ribosome-released nascent chains. Our results show that electrostatic interactions due to the negatively charged ribosomal surface play a role in the regulation of co-translational folding of nascent chain. Polymer theory calculations also support these results.