Evidence of functional protein dynamics from X-ray crystallographic ensembles.

It is widely recognized that representing a protein as a single static conformation is inadequate to describe the dynamics essential to the performance of its biological function. We contrast the amino acid displacements below and above the protein dynamical transition temperature, T(D)∼215K, of hen egg white lysozyme using X-ray crystallography ensembles that are analyzed by molecular dynamics simulations as a function of temperature. We show that measuring structural variations across an ensemble of X-ray derived models captures the activation of conformational states that are of functional importance just above T(D), and they remain virtually identical to structural motions measured at 300K. Our results highlight the ability to observe functional structural variations across an ensemble of X-ray crystallographic data, and that residue fluctuations measured in MD simulations at room temperature are in quantitative agreement with the experimental observable.

Statistics and physical origins of pK and ionization state changes upon protein-ligand binding.

This work investigates statistical prevalence and overall physical origins of changes in charge states of receptor proteins upon ligand binding. These changes are explored as a function of the ligand type (small molecule, protein, and nucleic acid), and distance from the binding region. Standard continuum solvent methodology is used to compute, on an equal footing, pK changes upon ligand binding for a total of 5899 ionizable residues in 20 protein-protein, 20 protein-small molecule, and 20 protein-nucleic acid high-resolution complexes. The size of the data set combined with an extensive error and sensitivity analysis allows us to make statistically justified and conservative conclusions: in 60% of all protein-small molecule, 90% of all protein-protein, and 85% of all protein-nucleic acid complexes there exists at least one ionizable residue that changes its charge state upon ligand binding at physiological conditions (pH = 6.5). Considering the most biologically relevant pH range of 4-8, the number of ionizable residues that experience substantial pK changes (DeltapK > 1.0) due to ligand binding is appreciable: on average, 6% of all ionizable residues in protein-small molecule complexes, 9% in protein-protein, and 12% in protein-nucleic acid complexes experience a substantial pK change upon ligand binding. These changes are safely above the statistical false-positive noise level. Most of the changes occur in the immediate binding interface region, where approximately one out of five ionizable residues experiences substantial pK change regardless of the ligand type. However, the physical origins of the change differ between the types: in protein-nucleic acid complexes, the pK values of interface residues are predominantly affected by electrostatic effects, whereas in protein-protein and protein-small molecule complexes, structural changes due to the induced-fit effect play an equally important role. In protein-protein and protein-nucleic acid complexes, there is a statistically significant number of substantial pK perturbations, mostly due to the induced-fit structural changes, in regions far from the binding interface.

How hot? Systematic convergence of the replica exchange method using multiple reservoirs.

We have devised a systematic approach to converge a replica exchange molecular dynamics simulation by dividing the full temperature range into a series of higher temperature reservoirs and a finite number of lower temperature subreplicas. A defined highest temperature reservoir of equilibrium conformations is used to help converge a lower but still hot temperature subreplica, which in turn serves as the high-temperature reservoir for the next set of lower temperature subreplicas. The process is continued until an optimal temperature reservoir is reached to converge the simulation at the target temperature. This gradual convergence of subreplicas allows for better and faster convergence at the temperature of interest and all intermediate temperatures for thermodynamic analysis, as well as optimizing the use of multiple processors. We illustrate the overall effectiveness of our multiple reservoir replica exchange strategy by comparing sampling and computational efficiency with respect to replica exchange, as well as comparing methods when converging the structural ensemble of the disordered Abeta(21-30) peptide simulated with explicit water by comparing calculated Rotating Overhauser Effect Spectroscopy intensities to experimentally measured values.

The influence of protein dynamics on the success of computational enzyme design.

We characterize the molecular dynamics of a previously described computational de novo designed enzyme optimized to perform a multistep retrol-aldol reaction when engineered into a TIM barrel protein scaffold. The molecular dynamics simulations show that the protein dynamics under physiological conditions of temperature and aqueous environment distorts the designed geometric factors of the substrate-enzyme reaction intermediates, such that catalysis is limited by the primary retrol-aldol step of proton abstraction from the covalently bound substrate and its interactions with a histidine-aspartate dyad. These results emphasize that computational enzyme designs will benefit from considerations of dynamical fluctuations when optimizing active site geometries.

Atomic level computational identification of ligand migration pathways between solvent and binding site in myoglobin.

Myoglobin is a globular protein involved in oxygen storage and transport. No consensus yet exists on the atomic level mechanism by which oxygen and other small nonpolar ligands move between the myoglobin's buried heme, which is the ligand binding site, and surrounding solvent. This study uses room temperature molecular dynamics simulations to provide a complete atomic level picture of ligand migration in myoglobin. Multiple trajectories--providing a cumulative total of 7 micros of simulation--are analyzed. Our simulation results are consistent with and tie together previous experimental findings. Specifically, we characterize: (i) Explicit full trajectories in which the CO ligand shuttles between the internal binding site and the solvent and (ii) pattern and structural origins of transient voids available for ligand migration. The computations are performed both in sperm whale myoglobin wild-type and in sperm whale V68F myoglobin mutant, which is experimentally known to slow ligand-binding kinetics. On the basis of these independent, but mutually consistent ligand migration and transient void computations, we find that there are two discrete dynamical pathways for ligand migration in myoglobin. Trajectory hops between these pathways are limited to two bottleneck regions. Ligand enters and exits the protein matrix in common identifiable portals on the protein surface. The pathways are located in the "softer" regions of the protein matrix and go between its helices and in its loop regions. Localized structural fluctuations are the primary physical origin of the simulated CO migration pathways inside the protein.

Structure and dynamics of the Abeta(21-30) peptide from the interplay of NMR experiments and molecular simulations.

We combine molecular dynamics simulations and new high-field NMR experiments to describe the solution structure of the Abeta(21-30) peptide fragment that may be relevant for understanding structural mechanisms related to Alzheimer's disease. By using two different empirical force-field combinations, we provide predictions of the three-bond scalar coupling constants ((3)J(H(N)H(alpha))), chemical-shift values, (13)C relaxation parameters, and rotating-frame nuclear Overhauser effect spectroscopy (ROESY) crosspeaks that can then be compared directly to the same observables measured in the corresponding NMR experiment of Abeta(21-30). We find robust prediction of the (13)C relaxation parameters and medium-range ROESY crosspeaks by using new generation TIP4P-Ew water and Amber ff99SB protein force fields, in which the NMR validates that the simulation yields both a structurally and dynamically correct ensemble over the entire Abeta(21-30) peptide. Analysis of the simulated ensemble shows that all medium-range ROE restraints are not satisfied simultaneously and demonstrates the structural diversity of the Abeta(21-30) conformations more completely than when determined from the experimental medium-range ROE restraints alone. We find that the structural ensemble of the Abeta(21-30) peptide involves a majority population (approximately 60%) of unstructured conformers, lacking any secondary structure or persistent hydrogen-bonding networks. However, the remaining minority population contains a substantial percentage of conformers with a beta-turn centered at Val24 and Gly25, as well as evidence of the Asp23 to Lys28 salt bridge important to the fibril structure. This study sets the stage for robust theoretical work on Abeta(1-40) and Abeta(1-42), for which collection of detailed NMR data on the monomer will be more challenging because of aggregation and fibril formation on experimental timescales at physiological conditions. In addition, we believe that the interplay of modern molecular simulation and high-quality NMR experiments has reached a fruitful stage for characterizing structural ensembles of disordered peptides and proteins in general.

A computational study of nucleosomal DNA flexibility.

Molecular dynamics simulations of the nucleosome core particle and its isolated DNA free in solution are reported. The simulations are based on the implicit solvent methodology and provide insights into the nature of large-scale structural fluctuations and flexibility of the nucleosomal DNA. In addition to the kinked regions previously identified in the x-ray structure of the nucleosome, the simulations support the existence of a biochemically identified distorted region of the DNA. Comparison of computed relative free energies shows that formation of the kinks is associated with little, if any, energy cost relative to a smooth, ideal conformation of the DNA superhelix. Isolated nucleosomal DNA is found to be considerably more flexible than expected for a 147 bp stretch of DNA based on its canonical persistence length of 500 A. Notably, the significant bending of the DNA observed in our simulations occurs without breaking of Watson-Crick bonds. The computed relative stability of bent conformations is sensitive to the ionic strength of the solution in the physiological range; the sensitivity suggests possible experiments that might provide further insights into the structural origins of the unusual flexibility of the DNA.