Biomedical subjects

Dusan Bratko

Publications and source records attributed to Dusan Bratko.

8 recordsLinked to original sources

PubMed2007

Molecular simulation of protein aggregation.

Computer simulation offers unique possibilities for investigating molecular-level phenomena difficult to probe experimentally. Drawing from a wealth of studies concerning protein folding, computational studies of protein aggregation are emerging. These studies have been successful in capturing aspects of aggregation known from experiment and are being used to refine experimental methods aimed at abating aggregation. Here we review molecular-simulation studies of protein aggregation conducted in our laboratory. Specific attention is devoted to issues with implications for biotechnology.

Binding Sites↗

PubMed2006

Effect of single-point sequence alterations on the aggregation propensity of a model protein.

Sequences of contemporary proteins are believed to have evolved through a process that optimized their overall fitness, including their resistance to deleterious aggregation. Biotechnological processing may expose therapeutic proteins to conditions that are much more conducive to aggregation than those encountered in a cellular environment. An important task of protein engineering is to identify alternative sequences that would protect proteins when processed at high concentrations without altering their native structure associated with specific biological function. Our computational studies exploit parallel tempering simulations of coarse-grained model proteins to demonstrate that isolated amino acid residue substitutions can result in significant changes in the aggregation resistance of the protein in a crowded environment while retaining protein structure in isolation. A thermodynamic analysis of protein clusters subject to competing processes of folding and association shows that moderate mutations can produce effects similar to those caused by changes in system conditions, including temperature, concentration, and solvent composition, that affect the aggregation propensity. The range of conditions where a protein can resist aggregation can therefore be tuned by sequence alterations, although the protein generally may retain its generic ability for aggregation.

Amino Acid Substitution↗

PubMed2005

Gas solubility in hydrophobic confinement.

Measured forces between apolar surfaces in water have often been found to be sensitive to exposure to atmospheric gases despite low gas solubilities in bulk water. This raises questions as to how significant gas adsorption is in hydrophobic confinement, whether it is conducive to water depletion at such surfaces, and ultimately if it can facilitate the liquid-to-gas phase transition in the confinement. Open Ensemble molecular simulations have been used here to determine saturated concentrations of atmospheric gases in water-filled apolar confinements as a function of pore width at varied gas fugacities. For paraffin-like confinements of widths barely exceeding the mechanical instability threshold (spinodal) of the liquid-to-vapor transition of confined water (aqueous film thickness between three and four molecular diameters), mean gas concentrations in the pore were found to exceed the bulk values by a factor of approximately 30 or approximately 15 in cases of N2 and CO2, respectively. At ambient conditions, this does not result in visible changes in the water density profile next to the surfaces. Whereas the barrier to capillary evaporation has been found to decrease in the presence of dissolved gas (Leung, K.; Luzar, A.; and Bratko, D. Phys. Rev. Lett. 2003, 90, 065502), gas concentrations much higher than those observed at normal atmospheric conditions would be needed to produce noticeable changes in the kinetics of capillary evaporation. In simulations, dissolved gas concentrations corresponding to fugacities above approximately 40 bar for N2, or approximately 2 bar for CO2, were required to trigger expulsion of water from a hydrocarbon slit as narrow as 1.4 nm. For nanosized pore widths corresponding to the mechanical instability threshold or above, no significant coupling between adsorption layers at opposing confinement walls was observed. This finding explains the approximately linear increase in gas solubility with inverse confinement width and the apparent validity of Henry's law in the pores over a broad fugacity range.

Adsorption↗

PubMed2005

Protein-folding landscapes in multichain systems.

Computational studies of proteins have significantly improved our understanding of protein folding. These studies are normally carried out by using chains in isolation. However, in many systems of practical interest, proteins fold in the presence of other molecules. To obtain insight into folding in such situations, we compare the thermodynamics of folding for a Miyazawa-Jernigan model 64-mer in isolation to results obtained in the presence of additional chains. The melting temperature falls as the chain concentration increases. In multichain systems, free-energy landscapes for folding show an increased preference for misfolded states. Misfolding is accompanied by an increase in interprotein interactions; however, near the folding temperature, the transition from folded chains to misfolded and associated chains is entropically driven. A majority of the most probable interprotein contacts are also native contacts, suggesting that native topology plays a role in early stages of aggregation.

Amino Acids↗

PubMed2005

Thermodynamics of folding and association of lattice-model proteins.

Closely related to the "protein folding problem" is the issue of protein misfolding and aggregation. Protein aggregation has been associated with the pathologies of nearly 20 human diseases and presents serious difficulties during the manufacture of pharmaceutical proteins. Computational studies of multiprotein systems have recently emerged as a powerful complement to experimental efforts aimed at understanding the mechanisms of protein aggregation. We describe the thermodynamics of systems containing two lattice-model 64-mers. A parallel tempering algorithm abates problems associated with glassy systems and the weighted histogram analysis method improves statistical quality. The presence of a second chain has a substantial effect on single-chain conformational preferences. The melting temperature is substantially reduced, and the increase in the population of unfolded states is correlated with an increase in interactions between chains. The transition from two native chains to a non-native aggregate is entropically favorable. Non-native aggregates receive approximately 25% of their stabilizing energy from intraprotein contacts not found in the lowest-energy structure. Contact maps show that for non-native dimers, nearly 50% of the most probable interprotein contacts involve pairs of residues that form native contacts, suggesting that a domain-swapping mechanism is involved in self-association.

Binding Sites↗

PubMed2005

The competition between protein folding and aggregation: off-lattice minimalist model studies.

Protein aggregation has been associated with a number of human diseases, and is a serious problem in the manufacture of recombinant proteins. Of particular interest to the biotechnology industry is deleterious aggregation that occurs during the refolding of proteins from inclusion bodies. As a complement to experimental efforts, computer simulations of multi-chain systems have emerged as a powerful tool to investigate the competition between folding and aggregation. Here we report results from Langevin dynamics simulations of minimalist model proteins. Order parameters are developed to follow both folding and aggregation. By mapping natural units to real units, the simulations are shown to be carried out under experimentally relevant conditions. Data pertaining to the contacts formed during the association process show that multiple mechanisms for aggregation exist, but certain pathways are statistically preferred. Kinetic data show that there are multiple time scales for aggregation, although most association events take place at times much shorter than those required for folding. Last, we discuss results presented here as a basis for future work aimed at rational design of mutations to reduce aggregation propensity, as well as for development of small-molecular weight refolding enhancers.

Amino Acid Sequence↗

PubMed2004

Effect of alcohols on aqueous lysozyme-lysozyme interactions from static light-scattering measurements.

Alcohols have been widely used as protein denaturants, precipitants and crystallization reagents. We have studied the effect of alcohols on aqueous hen-egg lysozyme self-interactions by measuring the osmotic second virial coefficient (B22) using static light scattering. Addition of alcohols increases B22, indicating stronger protein-protein repulsion or weaker attraction. For the monohydric alcohols used in this study (methanol, ethanol, 1-propanol, n-butanol, iso-butanol and trifluoroethanol), B22 for lysozyme reaches a common plateau at approximately 5% (v/v) alcohol, while glycerol increases B22 more than monohydric alcohols. For a 0.05 M NaCl hen-egg lysozyme solution at pH 7, B22 increases from 2.4 x 10(-4) to 4.7 x 10(-4) ml mol/g2 upon addition of monohydric alcohols and to 5.8 x 10(-4) ml mol/g2 upon addition of glycerol. We describe the alcohol effect using a simple model that supplements the DLVO theory with an additional alcohol-dependent term representing orientation-averaged hydrophobic interactions. In this model, the increased lysozyme repulsive forces in the presence of monohydric alcohols are interpreted in terms of adsorption of alcohol molecules on hydrophobic sites on the protein surface. This adsorption reduces attractive hydrophobic protein-protein interactions. A thicker lysozyme hydration layer in aqueous glycerol solution can explain the glycerol-increased lysozyme-lysozyme repulsion.

Alcohols↗

PubMed2003

Dynamics of capillary drying in water.

We use atomistic simulations to address the question when capillary evaporation of water confined in a hydrocarbonlike slit is kinetically viable. Activation barriers and absolute rates of evaporation are estimated using open ensemble Monte Carlo-umbrella sampling and molecular dynamics simulations. At ambient conditions, the evaporation rate in a water film four molecular diameters thick is found to be of the order 10(5)(nm(2) s)(-1), meaning that water readily evaporates. Films more than a few nanometers thick will persist in a metastable liquid state. Dissolved atmospheric gas molecules do not significantly decrease the activation barrier.

Journal Article↗