Determining the three-dimensional structure of myoglobin, the first solved structure of a protein, fundamentally changed the way protein function was understood. Even more revolutionary was the information that came afterward: protein dynamics play a critical role in biological functions. Therefore, understanding conformational dynamics is crucial to obtaining a more complete picture of protein evolution. We recently analyzed the evolution of different protein families including green fluorescent proteins (GFPs), β-lactamase inhibitors, and nuclear receptors, and we observed that the alteration of conformational dynamics through allosteric regulation leads to functional changes. Moreover, proteome-wide conformational dynamics analysis of more than 100 human proteins showed that mutations occurring at rigid residue positions are more susceptible to disease than flexible residue positions. These studies suggest that disease-associated mutations may impair dynamic allosteric regulations, leading to loss of function. Thus, in this study, we analyzed the conformational dynamics of the wild-type light chain subunit of human ferritin protein along with the neutral and disease forms. We first performed replica exchange molecular dynamics simulations of wild-type and mutants to obtain equilibrated dynamics and then used perturbation response scanning (PRS), where we introduced a random Brownian kick to a position and computed the fluctuation response of the chain using linear response theory. Using this approach, we computed the dynamic flexibility index (DFI) for each position in the chain for the wild-type and the mutants. DFI quantifies the resilience of a position to a perturbation and provides a flexibility/rigidity measurement for a given position in the chain. The DFI analysis reveals that neutral variants and the wild-type exhibit similar flexibility profiles in which experimentally determined functionally critical sites act as hinges in controlling the overall motion. However, disease mutations alter the conformational dynamic profile, making hinges more loose (i.e., softening the hinges), thus impairing the allosterically regulated dynamics.

Protein evolution is most commonly studied by analyzing related protein sequences and generating ancestral sequences through Bayesian and Maximum Likelihood methods, and/or by resurrecting ancestral proteins in the lab and performing ligand binding studies to determine function. Structural and dynamic evolution have largely been left out of molecular evolution studies. Here we incorporate both structure and dynamics to elucidate the molecular principles behind the divergence in the evolutionary path of the steroid receptor proteins. We determine the likely structure of three evolutionarily diverged ancestral steroid receptor proteins using the Zipping and Assembly Method with FRODA (ZAMF). Our predictions are within ∼2.7 Å all-atom RMSD of the respective crystal structures of the ancestral steroid receptors. Beyond static structure prediction, a particular feature of ZAMF is that it generates protein dynamics information. We investigate the differences in conformational dynamics of diverged proteins by obtaining the most collective motion through essential dynamics. Strikingly, our analysis shows that evolutionarily diverged proteins of the same family do not share the same dynamic subspace, while those sharing the same function are simultaneously clustered together and distant from those, that have functionally diverged. Dynamic analysis also enables those mutations that most affect dynamics to be identified. It correctly predicts all mutations (functional and permissive) necessary to evolve new function and ∼60% of permissive mutations necessary to recover ancestral function.

Although proteins are a fundamental unit in biology, the mechanism by which proteins fold into their native state is not well understood. In this work, we explore the assembly of secondary structure units via geometric constraint-based simulations and the effect of refinement of assembled structures using reservoir replica exchange molecular dynamics. Our approach uses two crucial features of these methods: i), geometric simulations speed up the search for nativelike topologies as there are no energy barriers to overcome; and ii), molecular dynamics identifies the low free energy structures and further refines these structures toward the actual native conformation. We use eight alpha-, beta-, and alpha/beta-proteins to test our method. The geometric simulations of our test set result in an average RMSD from native of 3.7 A and this further reduces to 2.7 A after refinement. We also explore the question of robustness of assembly for inaccurate (shifted and shortened) secondary structure. We find that the RMSD from native is highly dependent on the accuracy of secondary structure input, and even slightly shifting the location of secondary structure along the amino acid sequence can lead to a rapid decrease in RMSD to native due to incorrect packing.

Human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/ AIDS) currently represents the fourth leading cause of death worldwide and has claimed the lives of over 25 million people since 1981. To combat this infectious disease, a wide range of drugs has been discovered, inhibiting various stages of the retroviral life cycle. The most popular target is the HIV-1 reverse transcriptase (RT) enzyme, which is required to convert retroviral RNA into DNA, and for which substantial high-resolution structural data exist. Here, we focus on the non-nucleoside RT inhibitor (NNRTI) class of compounds, which confer high specificity and block DNA polymerization in an allosteric fashion. Despite the FDA-approval of four NNRTIs to date, the side-effects of drug toxicity and the emergence of drug-resistance mutations demand further drug discovery endeavors. To this end, we have performed a virtual screening study of the RT enzyme, with the aim of discovering novel NNRTI lead compounds from the National Cancer Institute (NCI) library. To take into account the conformational flexibility of the protein, we have screened minilibraries of NCI compounds against diverse ensembles of RT structures. Firstly, we use a traditional, experimental source of structures: x-ray crystallography. Next, in an effort to expose novel conformations of the binding site that might be missed experimentally, we use a theoretical source: molecular dynamics (MD) simulation. To achieve this, we have carried out all-atom, explicit solvent MD simulation of the RT enzyme, complexed with a variety of NNRTIs. By integrating the results from the crystallographic and MD ("relaxed") ensemble screens, we compile a set of the most promising candidate compounds for experimental testing.

re-equilibration of a chemical system following an instantaneous increase in temperature induced by a laser pulse tuned to an infrared water band. The reequilibration results in changes in the concentration of the species involved, and the transient changes are characterized using spectroscopic probes. To investigate the conformational changes associated with the binding of oxamate we studied the LDH from wild type cells as well as those from various single tryptophan mutants. These mutants were created by first replacing all tryptophans with tyrosine in wild type bsLDH to create a tryptophan-less template, followed by reintroduction of a single tryptophan at strategic sites in the protein. We probed the fluorescence emission of NADH in wild type and mutant bsLDH to report on the time evolution of the changes within the NADH environment over 100ms to 3ms time scale. Transients collected were then correlated to those resulting from a probe of tryptophan emissions. The results were then analyzed based on a plausible kinetic model. A comprehensive picture of the dynamics of ligand binding and Michaelis complex formation in bsLDH is obtained from the various structural reporters.