Marimuthu Krishnan

Methyl group dynamics and the onset of anharmonicity in myoglobin

The role of methyl groups in the onset of low-temperature anharmonic dynamics in a crystalline protein at low temperature is investigated using atomistic molecular dynamics (MD) simulation. Anharmonicity appears at approximately 150 K, far below the much-studied solvent-activated dynamical transition at approximately 220 K. A significant fraction of methyl groups exhibit nanosecond time scale rotational jump diffusion at 150 K. The splitting and shift in peak position of both the librational band (around 100 cm(-1)) and the torsional band (around 270-300 cm(-1)) also differ significantly among methyl groups, depending on the local environment. The simulation results provide no evidence for a correlation between methyl dynamics and solvent exposure, consistent with the hydration-independence of the low-temperature anharmonic dynamics observed in neutron scattering experiments. The calculated proton mean-square fluctuation and methyl NMR order parameters show a systematic nonlinear dependence on the rotational barrier which can be described using model functions. The methyl groups that exhibit many rotational excitations are located near xenon cavities, suggesting that cavities in proteins act as activation centers of anharmonic dynamics. The dynamic heterogeneity and the environmental sensitivity of motional parameters and low-frequency spectral bands of CH(3) groups found here suggest that methyl dynamics may be used as a probe to investigate the relation between low-energy structural fluctuations and packing defects in proteins.

Response of Small-Scale, Methyl Rotors to Protein−Ligand Association: A Simulation Analysis of Calmodulin−Peptide Binding

Changes in the free energy barrier (DeltaE), entropy, and motional parameters associated with the rotation of methyl groups in a protein (calmodulin (CaM)) on binding a ligand (the calmodulin-binding domain of smooth-muscle myosin (smMLCKp)) are investigated using molecular dynamics simulation. In both the bound and uncomplexed forms of CaM, the methyl rotational free energy barriers follow skewed-Gaussian distributions that are not altered significantly upon ligand binding. However, site-specific perturbations are found. Around 11% of the methyl groups in CaM exhibit changes in DeltaE greater than 0.7 kcal/mol on binding. The rotational entropies of the methyl groups exhibit a nonlinear dependence on DeltaE. The relations are examined between motional parameters (the methyl rotational NMR order parameter and the relaxation time) and DeltaE. Low-barrier methyl group rotational order parameters deviate from ideal tetrahedrality by up to approximately 20%. There is a correlation between rotational barrier changes and proximity to the protein-peptide binding interface. Methyl groups that exhibit large changes in DeltaE are found to report on elements in the protein undergoing structural change on binding.

Reconstruction of Protein Side-Chain Conformational Free Energy Surfaces From NMR-Derived Methyl Axis Order Parameters

An analytical approach is developed for reconstructing site-specific methyl-bearing protein side-chain conformational energy surfaces from NMR methyl axis order parameters (O(axis)(2)). Application of an enhanced sampling algorithm (adaptive biasing force) to molecular dynamics simulation of a protein, calcium-bound calmodulin, reveals a nonlinear correlation between O(axis)(2) and the populations of rotamer states of protein side-chains, permitting the rotamer populations to be extracted directly from O(axis)(2). The analytical approach yields side-chain conformational distributions that are in excellent agreement with those obtained from the enhanced-sampling MD results.

Instantaneous Normal Modes and the Protein Glass Transition

In the instantaneous normal mode method, normal mode analysis is performed at instantaneous configurations of a condensed-phase system, leading to modes with negative eigenvalues. These negative modes provide a means of characterizing local anharmonicities of the potential energy surface. Here, we apply instantaneous normal mode to analyze temperature-dependent diffusive dynamics in molecular dynamics simulations of a small protein (a scorpion toxin). Those characteristics of the negative modes are determined that correlate with the dynamical (or glass) transition behavior of the protein, as manifested as an increase in the gradient with T of the average atomic mean-square displacement at approximately 220 K. The number of negative eigenvalues shows no transition with temperature. Further, although filtering the negative modes to retain only those with eigenvectors corresponding to double-well potentials does reveal a transition in the hydration water, again, no transition in the protein is seen. However, additional filtering of the protein double-well modes, so as to retain only those that, on energy minimization, escape to different regions of configurational space, finally leads to clear protein dynamical transition behavior. Partial minimization of instantaneous configurations is also found to remove nondiffusive imaginary modes. In summary, examination of the form of negative instantaneous normal modes is shown to furnish a physical picture of local diffusive dynamics accompanying the protein glass transition.

Hidden Regularity and Universal Classification of Fast Side Chain Motions in Proteins

Proteins display characteristic dynamical signatures that appear to be universal across all proteins regardless of topology and size. Here, we systematically characterize the universal features of fast side chain motions in proteins by examining the conformational energy surfaces of individual residues obtained using enhanced sampling molecular dynamics simulation (618 free energy surfaces obtained from 0.94 μs MD simulation). The side chain conformational free energy surfaces obtained using the adaptive biasing force (ABF) method for a set of eight proteins with different molecular weights and secondary structures are used to determine the methyl axial NMR order parameters (O(axis)(2)), populations of side chain rotamer states (ρ), conformational entropies (S(conf)), probability fluxes, and activation energies for side chain inter-rotameric transitions. The free energy barriers separating side chain rotamer states range from 0.3 to 12 kcal/mol in all proteins and follow a trimodal distribution with an intense peak at ~5 kcal/mol and two shoulders at ~3 and ~7.5 kcal/mol, indicating that some barriers are more favored than others by proteins to maintain a balance between their conformational stability and flexibility. The origin and the influences of the trimodal barrier distribution on the distribution of O(axis)(2) and the side chain conformational entropy are discussed. A hierarchical grading of rotamer states based on the conformational free energy barriers, entropy, and probability flux reveals three distinct classes of side chains in proteins. A unique nonlinear correlation is established between O(axis)(2) and the side chain rotamer populations (ρ). The apparent universality in O(axis)(2) versus ρ correlation, trimodal barrier distribution, and distinct characteristics of three classes of side chains observed among all proteins indicates a hidden regularity (or commonality) in the dynamical heterogeneity of fast side chain motions in proteins.

Three Entropic Classes of Side Chain in a Globular Protein

The relationship between the NMR methyl group axial order parameter and the side chain conformational entropy is investigated in inhibitor-bound and apo human HIV protease using molecular dynamics simulation. Three distinct entropic classes of methyl-bearing side chains, determined by the topological distance of the methyl group from the protein backbone (i.e., the number of χ-bonds between the Cα and the carbon of the CH3 group), are revealed by atomistic trajectory analyses performed in the local frame of reference of individual methyl probes. The results demonstrate that topologically equivalent methyl groups experience similar nonbonded microenvironments regardless of the type of residues to which they are attached. Similarly, methyl groups that belong to the same side chain but that are not topologically equivalent exhibit different thermodynamic and dynamic properties. The two-parameter classification (based upon entropy and methyl axial order parameter) of side chains described here permits improved estimates of the conformational entropies of proteins from NMR motional parameters.

Direct Determination of Site-Specific Noncovalent Interaction Strengths of Proteins from NMR-Derived Fast Side Chain Motional Parameters

A novel approach to accurately determine residue-specific noncovalent interaction strengths (ξ) of proteins from NMR-measured fast side chain motional parameters (Oaxis2) is presented. By probing the environmental sensitivity of side chain conformational energy surfaces of individual residues of a diverse set of proteins, the microscopic connections between ξ, Oaxis2, conformational entropy (Sconf), conformational barriers, and rotamer stabilities established here are found to be universal among proteins. The results reveal that side chain flexibility and conformational entropy of each residue decrease with increasing ξ and that for each residue type there exists a critical range of ξ, determined primarily by the mean side chain conformational barriers, within which flexibility of any residue can be reversibly tuned from highly flexible (with Oaxis2 ∼ 0) to highly restricted (with Oaxis2 ∼ 1) by increasing ξ by ∼3 kcal/mol. Beyond this critical range of ξ, both side chain flexibility and conformational entropy are insensitive to ξ. The interrelationships between conformational dynamics, conformational entropy, and noncovalent interactions of protein side chains established here open up new avenues to probe perturbation-induced (for example, ligand-binding, temperature, pressure) changes in fast side chain dynamics and thermodynamics of proteins by comparing their conformational energy surfaces in the native and perturbed states.

Structure and Dynamics of Biological Systems: Integration of Neutron Scattering with Computer Simulation

The combination of molecular dynamics simulation and neutron scattering techniques has emerged as a highly synergistic approach to elucidate the atomistic details of the structure, dynamics, and functions of biological systems. Simulation models can be tested by calculating neutron scattering structure factors and comparing the results directly with experiments. If the scattering profiles agree, the simulations can be used to provide a detailed decomposition and interpretation of the experiments, and if not, the models can be rationally adjusted. Comparison with neutron experiment can be made at the level of the scattering functions or, less directly, of structural and dynamical quantities derived from them. Here, we examine the combination of simulation and experiment in the interpretation of SANS and inelastic scattering experiments on the structure and dynamics of proteins and other biopolymers.

Protein Dynamical Transition: Role of Methyl Dynamics and Local Diffusion

The temperature‐dependent protein dynamical transition is investigated using the Instanteous Normal mode analysis (INM) and molecular dynamics (MD) simulation of crystalline myoglobin and Toxin II. The onset of anharmonic dynamics in myoglobin is observed at 150 K, far below the much‐studied solvent‐activated dynamical transition at 220 K. A significant fraction of methyl groups exhibit nanosecond anharmonic rotational jump diffusion at 150 K indicating the essential role of methyl dynamics in the low‐temperature onset of anharmonic protein dynamics. The methyl groups that exhibit many rotational excitations are located near xenon cavities, suggesting that cavities in proteins act as activation centers of anharmonic dynamics. INM analysis of Toxin II indicates the presence of non‐zero barrier‐crossing, diffusive degrees of freedom accessible to the protein below the dynamical transition. The number of these diffusive degrees of freedom increases abruptly at the dynamical transition. In summary, the presen...

Molecular Mechanism, Dynamics, and Energetics of Protein-Mediated Dinucleotide Flipping in a Mismatched DNA: A Computational Study of the RAD4-DNA Complex

DNA damage alters genetic information and adversely affects gene expression pathways leading to various complex genetic disorders and cancers. DNA repair proteins recognize and rectify DNA damage and mismatches with high fidelity. A critical molecular event that occurs during most protein-mediated DNA repair processes is the extrusion of orphaned bases at the damaged site facilitated by specific repairing enzymes. The molecular-level understanding of the mechanism, dynamics, and energetics of base extrusion is necessary to elucidate the molecular basis of protein-mediated DNA damage repair. The present article investigates the molecular mechanism of dinucleotide extrusion in a mismatched DNA (containing a stretch of three contiguous thymidine-thymidine base pairs) facilitated by Radiation sensitive 4 (RAD4), a key DNA repair protein, on an atom-by-atom basis using molecular dynamics (MD) and umbrella-sampling (US) simulations. Using atomistic models of RAD4-free and RAD4-bound mismatched DNA, the free energy profiles associated with extrusion of mismatched partner bases are determined for both systems. The mismatched bases adopted the most stable intrahelical conformation, and their extrusion was unfavorable in RAD4-free mismatched DNA due to the presence of prohibitively high barriers (>12.0 kcal/mol) along the extrusion pathways. Upon binding of RAD4 to the DNA, the global free energy minimum is shifted to the extrahelical state indicating the key role of RAD4-DNA interactions in catalyzing the dinucleotide base extrusion in the DNA-RAD4 complex. The critical residues of RAD4 contributing to the conformational stability of the mismatched bases are identified, and the energetics of insertion of a β-hairpin of RAD4 into the DNA duplex is examined. The conformational energy landscape-based mechanistic insight into RAD4-mediated base extrusion provided here may serve as a useful baseline to understand the molecular basis of xeroderma pigmentosum C (XPC)-mediated DNA damage repair in humans.