Biomedical subjects

Troy Cellmer

Publications and source records attributed to Troy Cellmer.

7 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

Kinetic studies of protein L aggregation and disaggregation.

We have investigated the aggregation of protein L in 25% (vol/vol) TFE and 10 mM HCl. Under both conditions, aggregates adopt a fibrillar structure and bind dyes Congo Red and Thioflavin T consistent with the presence of amyloid fibrils. The kinetics of aggregation in 25% TFE suggest a linear-elongation mechanism with critical nucleus size of either two or three monomers. Aggregation kinetics in 10 mM HCl show a prolonged lag phase prior to a rapid increase in aggregation. The lag phase is time-dependent, but the time dependence can be eliminated by the addition of pre-formed seeds. Disaggregation studies show that for aggregates formed in TFE, aggregate stability is a strong function of aggregate age. For example, after 200 min of aggregation, 40% of the aggregation reaction is irreversible, while after 3 days over 60% is irreversible. When the final concentration of the denaturant, TFE, is reduced from 5% to 0, the amount of reversible aggregation doubles. Disaggregation studies of aggregates formed in TFE and 10 mM HCl reveal a complicated effect of pH on aggregate stability.

Amyloidosis↗

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

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

Interactions of lysozyme in guanidinium chloride solutions from static and dynamic light-scattering measurements.

The interactions of partially unfolded proteins provide insight into protein folding and protein aggregation. In this work, we studied partially unfolded hen egg lysozyme interactions in solutions containing up to 7 M guanidinium chloride (GdnHCl). The osmotic second virial coefficient (B(22)) of lysozyme was measured using static light scattering in GdnHCl aqueous solutions at 20 degrees C and pH 4.5. B(22) is positive in all solutions, indicating repulsive protein-protein interactions. At low GdnHCl concentrations, B(22) decreases with rising ionic strength: in the absence of GdnHCl, B(22) is 1.1 x 10(-3) mLmol/g(2), decreasing to 3.0 x 10(-5) mLmol/g(2) in the presence of 1 M GdnHCl. Lysozyme unfolds in solutions at GdnHCl concentrations higher than 3 M. Under such conditions, B(22) increases with ionic strength, reaching 8.0 x 10(-4) mLmol/g(2) at 6.5 M GdnHCl. Protein-protein hydrodynamic interactions were evaluated from concentration-dependent diffusivity measurements, obtained from dynamic light scattering. At moderate GdnHCl concentrations, lysozyme interparticle interactions are least repulsive and hydrodynamic interactions are least attractive. The lysozyme hydrodynamic radius was calculated from infinite-dilution diffusivity and did not change significantly during protein unfolding. Our results contribute toward better understanding of protein interactions of partially unfolded states in the presence of a denaturant; they may be helpful for the design of protein refolding processes that avoid protein aggregation.

Animals↗

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↗