Biology subjects

Denesyuk, N. A.

Publications and source records attributed to Denesyuk, N. A..

5 recordsLinked to original sources

bioRxiv2020

Theory and simulations for crowding-induced changes in stability of proteins with applications to {lambda} repressor

Experiments and theories have shown that when steric interactions between crowding particles and proteins are dominant, which give rise to Asakura-Oosawa depletion forces, then the stabilities of the proteins increase compared to the infinite dilution case. We show using theoretical arguments that the crowder volume fraction ({Phi}C) dependent increase in the melting temperature of globular proteins, [Formula], where [Formula]. The effective Flory exponent,{nu} eff, relates the radius of gyration in the unfolded state to the number of amino acid residues in the protein. We derive the bound 1.25 [≤] [≤] 2.0. The theoretical predictions are confirmed using molecular simulations of {lambda} repressor in the presence of spherical crowding particles. Analyses of previous simulations and experiments confirm the predicted theoretical bound for . We show that the non-specific attractions between crowding particles and amino acid residues have to be substantial to fully negate the enhanced protein stabilities due to intra protein attractive Asakura-Oosawa (AO) depletion potential. Using the findings, we provide an alternate explanation for the very modest (often less than 0.5 Kcal/mol) destabilization in certain proteins in the cellular milieu. Cellular environment is polydisperse containing large and small crowding agents. AO arguments suggest that proteins would be localized between large (sizes exceeding that of the proteins) crowders, which are predicted to have negligible effect on stability. In vitro experiments containing mixtures of crowding particles could validate or invalidate the predictions.

biophysics↗

bioRxiv2020

Molecular Simulations of Mg2+-induced Folding of the Central Domain of the 16S Ribosomal RNA

Both the small and large subunits of the ribosome, the molecular machine that synthesizes proteins, are complexes of ribosomal RNAs (rRNAs) and a number of proteins. In bacteria, the small subunit has a single 16S rRNA whose folding is the first step in its assembly. The central domain of the 16S rRNA folds independently, driven either by Mg2+ ions or by interaction with ribosomal proteins. In order to provide a quantitative description of ion-induced folding of the [~]350 nucleotide rRNA, we carried out extensive coarse-grained molecular simulations spanning Mg2+ concentration between 0-30 mM. The Mg2+ dependence of the radius of gyration shows that globally the rRNA folds cooperatively. Surprisingly, various structural elements order at different Mg2+ concentrations, indicative of the heterogeneous assembly even within a single domain of the rRNA. Binding of Mg2+ ions is highly specific, with successive ion condensation resulting in nucleation of tertiary structures. We also predict the Mg2+-dependent protection factors, measurable in hydroxyl radical footprinting experiments, which corroborate the specificity of Mg2+-induced folding. The simulations, which agree quantitatively with several experiments on the folding of a three-way junction, show that its folding is preceded by formation of other tertiary contacts in the central junction. Our work provides a starting point in simulating the early events in the assembly of the small subunit of the ribosome. O_TEXTBOXSignificance StatementRibosomes are complexes between ribosomal RNA (rRNA) and a number of proteins. Because ribosome assembly begins with rRNA folding, we simulated the molecular details of Mg2+-driven folding of the central domain of the bacterial rRNA. Good agreement with experiments on the folding of the three-way junction in the center of the rRNA validates the model. Coupling of rRNA folding and Mg2+ binding shows that ions interact with rRNA segments in a coordinated manner. The shape of rRNA changes from a sphere in the unfolded state to a prolate ellipsoid at high Mg2+ concentration, which is the opposite of what transpires when a globular protein folds. Our study pro-vides the needed framework for undertaking ion-driven folding of large RNA molecules. C_TEXTBOX

biophysics↗

bioRxiv2019

Ion Condensation onto Ribozyme is Site-Specific and Fold-Dependent

The highly charged RNA molecules, with each phosphate carrying a single negative charge, cannot fold into well-defined architectures with tertiary interactions, in the absence of ions. For ribozymes, divalent cations are known to be more efficient than monovalent ions in driving them to a compact state although often Mg2+ ions are needed for catalytic activity. Therefore, how ions interact with RNA is relevant in understanding RNA folding. It is often thought that the most of the ions are territorially and non-specifically bound to the RNA, as predicted by the counterion condensation (CIC) theory. Here, we show using simulations of Azoarcus ribozyme, based on an accurate coarse-grained Three Site Interaction (TIS) model, with explicit divalent and monovalent cations, that ion condensation is highly specific and depends on the nucleotide position. The regions with high coordination between the phosphate groups and the divalent cations are discernible at very low concentrations when the ribozyme does not form tertiary interactions. Surprisingly, these regions also contain the secondary structural elements that nucleate subsequently in the self-assembly of RNA, implying that ion condensation is determined by the architecture of the folded state. These results are in sharp contrast to interactions of ions (monovalent and divalent) with rigid charged rods in which ion condensation is uniform and position independent. The differences are explained in terms of the dramatic non-monotonic shape fluctuations in the ribozyme as it folds with increasing Mg2+ or Ca2+ concentration.

biophysics↗

bioRxiv2018

Frictional effects on RNA folding: Speed limit and Kramers turnover

We investigated frictional effects on the folding rates of a human Telomerase hairpin (hTR HP) and H-type pseudoknot from the Beet Western Yellow Virus (BWYV PK) using simulations of the Three Interaction Site (TIS) model for RNA. The heat capacity from TIS model simulations, calculated using temperature replica exchange simulations, reproduces nearly quantitatively the available experimental data for the hTR HP. The corresponding results for BWYV PK serve as predictions. We calculated the folding rates (kFs) from more than 100 folding trajectories for each value of the solvent viscosity ({eta}) at a fixed salt concentration of 200 mM. Using the theoretical estimate ([Formula] where N is number of nucleotides) for folding free energy barrier, kF data for both the RNAs are quantitatively fit using one dimensional Kramers theory with two parameters specifying the curvatures in the unfolded basin and the barrier top. In the high-friction regime ({eta} {gtrsim} 10-5 Pa{middle dot}s), for both HP and PK, kFs decrease as 1/{eta} whereas in the low friction regime kFs increase as{eta} increases, leading to a maximum folding rate at a moderate viscosity (~ 10-6 Pa{middle dot}s), which is the Kramers turnover. From the fits, we find that the speed limit to RNA folding at water viscosity is between (1 - 4)s, which is in accord with our previous theoretical prediction as well as results from several single molecule experiments. Both the RNA constructs fold by parallel pathways. Surprisingly, we find that the flux through the pathways could be altered by changing solvent viscosity, a prediction that is more easily testable in RNA than proteins.

biophysics↗

bioRxiv2018

Molecular simulations of the monovalent-ion dependent Folding Thermodynamics of RNA

How ions affect RNA folding thermodynamics and kinetics is an important but a vexing problem that remains unsolved. Experiments have shown that the free energy change, {Delta}G(c), of RNA upon folding varies with the salt concentration (c) as, {Delta}G(c) = kc ln c + const, where the coefficient kc is proportional to the difference in the uptake of ions (ion preferential coefficient), {Delta}{Gamma}, between the folded and unfolded states. We performed simulations of a coarse-grained model, by modeling electrostatic interactions implicitly and with explicit representation of ions, to elucidate the molecular underpinnings of the relationship between folding free energy and ion preferential coefficient. Without any input from experiments, the simulations quantitatively reproduce the heat capacity for the -1 frame shifting pseudoknot (PK) from Beet Western Yellow Virus, thus validating the model. We show that {Delta}G(c) calculated directly from {Delta}{Gamma} varies linearly with ln c (c < 0.2M), for a hairpin and the PK, thus demonstrating a molecular link between the two quantities for RNA molecules that undergo substantial conformational changes during folding. Explicit ion simulations also show the linear dependence of {Delta}G(c) on ln c at all c with kc = 2kBT, except that {Delta}G(c) values are shifted by about 2 kcal/mol higher than experiments at all salt concentrations. The discrepancy is due to an underestimate the {Gamma} values for both the folded and unfolded states, while giving accurate values for {Delta}{Gamma}. The predictions for the salt dependence of {Delta}{Gamma} are amenable to test using single molecule pulling experiments. Our simulations, representing a significant advance in quantitatively describing ion effects in RNA, show that the framework provided here can be used to obtain accurate thermodynamics of RNA folding.

biophysics↗