Eric J. Sorin

Direct calculation of the binding free energies of FKBP ligands

Direct calculations of the absolute free energies of binding for eight ligands to FKBP protein were performed using the Fujitsu BioServer massively parallel computer. Using the latest version of the general assisted model building with energy refinement (AMBER) force field for ligand model parameters and the Bennett acceptance ratio for computing free-energy differences, we obtained an excellent linear fit between the calculated and experimental binding free energies. The rms error from a linear fit is 0.4 kcal/mol for eight ligand complexes. In comparison with a previous study of the binding energies of these same eight ligand complexes, these results suggest that the use of improved model parameters can lead to more predictive binding estimates, and that these estimates can be obtained with significantly less computer time than previously thought. These findings make such direct methods more attractive for use in rational drug design.

Equilibrium conformational dynamics in an RNA tetraloop from massively parallel molecular dynamics

Conformational equilibrium within the ubiquitous GNRA tetraloop motif was simulated at the ensemble level, including 10 000 independent all-atom molecular dynamics trajectories totaling over 110 micros of simulation time. This robust sampling reveals a highly dynamic structure comprised of 15 conformational microstates. We assemble a Markov model that includes transitions ranging from the nanosecond to microsecond timescales and is dominated by six key loop conformations that contribute to fluctuations around the native state. Mining of the Protein Data Bank provides an abundance of structures in which GNRA tetraloops participate in tertiary contact formation. Most predominantly observed in the experimental data are interactions of the native loop structure within the minor groove of adjacent helical regions. Additionally, a second trend is observed in which the tetraloop assumes non-native conformations while participating in multiple tertiary contacts, in some cases involving multiple possible loop conformations. This tetraloop flexibility can act to counterbalance the energetic penalty associated with assuming non-native loop structures in forming tertiary contacts. The GNRA motif has thus evolved not only to readily participate in simple tertiary interactions involving native loop structure, but also to easily adapt tetraloop secondary conformation in order to participate in larger, more complex tertiary interactions.

Empirical force‐field assessment: The interplay between backbone torsions and noncovalent term scaling

The kinetic and thermodynamic aspects of the helix‐coil transition in polyalanine‐based peptides have been studied at the ensemble level using a distributed computing network. This study builds on a previous report, which critically assessed the performance of several contemporary force fields in reproducing experimental measurements and elucidated the complex nature of helix‐coil systems. Here we consider the effects of modifying backbone torsions and the scaling of noncovalent interactions. Although these elements determine the potential of mean force between atoms separated by three covalent bonds (and thus largely determine the local conformational distributions observed in simulation), we demonstrate that the interplay between these factors is both complex and force field dependent. We quantitatively assess the heliophilicity of several helix‐stabilizing potentials as well as the changes in heliophilicity resulting from such modifications, which can “make or break” the accuracy of a given force field, and our findings suggests that future force field development may need to better consider effect that vary with peptide length. This report also serves as an example of the utility of distributed computing in analyzing and improving upon contemporary force fields at the level of absolute ensemble equilibrium, the next step in force field development.

Molecular simulation of multistate peptide dynamics: A comparison between microsecond timescale sampling and multiple shorter trajectories

Molecular dynamics simulations of the RN24 peptide, which includes a diverse set of structurally heterogeneous states, are carried out in explicit solvent. Two approaches are employed and compared directly under identical simulation conditions. Specifically, we examine sampling by two individual long trajectories (microsecond timescale) and many shorter (MS) uncoupled trajectories. Statistical analysis of the structural properties indicates a qualitative agreement between these approaches. Microsecond timescale sampling gives large uncertainties on most structural metrics, while the shorter timescale of MS simulations results in slight structural memory for beta‐structure starting states. Additionally, MS sampling detects numerous transitions on a relatively short timescale that are not observed in microsecond sampling, while long simulations allow for detection of a few transitions on significantly longer timescales. A correlation between the complex free energy landscape and the kinetics of the equilibrium is highlighted by principal component analysis on both simulation sets. This report highlights the increased precision of the MS approach when studying the kinetics of complex conformational change, while revealing the complementary insight and qualitative agreement offered by far fewer individual simulations on significantly longer timescales.

Evaluating Molecular Mechanical Potentials for Helical Peptides and Proteins

Multiple variants of the AMBER all-atom force field were quantitatively evaluated with respect to their ability to accurately characterize helix-coil equilibria in explicit solvent simulations. Using a global distributed computing network, absolute conformational convergence was achieved for large ensembles of the capped A21 and Fs helical peptides. Further assessment of these AMBER variants was conducted via simulations of a flexible 164-residue five-helix-bundle protein, apolipophorin-III, on the 100 ns timescale. Of the contemporary potentials that had not been assessed previously, the AMBER-99SB force field showed significant helix-destabilizing tendencies, with beta bridge formation occurring in helical peptides, and unfolding of apolipophorin-III occurring on the tens of nanoseconds timescale. The AMBER-03 force field, while showing adequate helical propensities for both peptides and stabilizing apolipophorin-III, (i) predicts an unexpected decrease in helicity with ALA→ARG+ substitution, (ii) lacks experimentally observed 310 helical content, and (iii) deviates strongly from average apolipophorin-III NMR structural properties. As is observed for AMBER-99SB, AMBER-03 significantly overweighs the contribution of extended and polyproline backbone configurations to the conformational equilibrium. In contrast, the AMBER-99φ force field, which was previously shown to best reproduce experimental measurements of the helix-coil transition in model helical peptides, adequately stabilizes apolipophorin-III and yields both an average gyration radius and polar solvent exposed surface area that are in excellent agreement with the NMR ensemble.

Ensemble simulations: folding, unfolding and misfolding of a high-efficiency frameshifting RNA pseudoknot

Abstract Massive all-atom molecular dynamics simulations were conducted across a distributed computing network to study the folding, unfolding, misfolding and conformational plasticity of the high-efficiency frameshifting double mutant of the 26 nt potato leaf roll virus RNA pseudoknot. Our robust sampling, which included over 40 starting structures spanning the spectrum from the extended unfolded state to the native fold, yielded nearly 120 μs of cumulative sampling time. Conformational microstate transitions on the 1.0 ns to 10.0 μs timescales were observed, with post-equilibration sampling providing detailed representations of the conformational free energy landscape and the complex folding mechanism inherent to the pseudoknot motif. Herein, we identify and characterize two alternative native structures, three intermediate states, and numerous misfolded states, the latter of which have not previously been characterized via atomistic simulation techniques. While in line with previous thermodynamics-based models of a general RNA folding mechanism, our observations indicate that stem-strand-sequence-separation may serve as an alternative predictor of the order of stem formation during pseudoknot folding. Our results contradict a model of frameshifting based on structural rigidity and resistance to mechanical unfolding, and instead strongly support more recent studies in which conformational plasticity is identified as a determining factor in frameshifting efficiency.

Chapter 8:Computer Simulations of Protein Folding

Computer simulation holds great promise to significantly complement experiment as a tool for biological and biophysical characterization. Simulations offer the promise of atomic spatial detail with femtosecond temporal resolution. However, the application of computational methodology has been greatl...

Ensemble Molecular Dynamics of a Protein-Ligand Complex: Residual Inhibitor Entropy Enhances Drug Potency in Butyrylcholinesterase

Butyrylcholinesterase is a key enzyme that catalyzes the hydrolysis of the neurotransmitter acetylcholine and shows an increased activity in patients suffering from Alzheimers disease (AD), making this enzyme a primary target in treating AD. Central to this problem, and to similar scenarios involving biomolecular recognition, is our understanding of the nature of the protein-ligand complex. The butyrylcholinesterase enzyme was studied via all-atom, explicit solvent, ensemble molecular dynamics simulations sans inhibitor and in the presence of three dialkyl phenyl phosphate inhibitors of known potency to a cumulative sampling of over 40 μs. Following the relaxation of these ensembles to conformational equilibria, binding modes for each inhibitor were identified. While classical models, which assume significant reduction in protein and ligand conformational entropies, continue to be favored in contemporary studies, our observations contradict those assumptions: bound ligands occupy many conformational states, thereby stabilizing the complex, while also promoting protein flexibility.