Peptide conformational preferences in osmolyte solutions: transfer free energies of decaalanine.

The nature in which the protecting osmolyte trimethylamine N-oxide (TMAO) and the denaturing osmolyte urea affect protein stability is investigated, simulating a decaalanine peptide model in multiple conformations of the denatured ensemble. Binary solutions of both osmolytes and mixed osmolyte solutions at physiologically relevant concentrations of 2:1 (urea:TMAO) are studied using standard molecular dynamics simulations and solvation free energy calculations. Component analysis reveals the differences in the importance of the van der Waals (vdW) and electrostatic interactions for protecting and denaturing osmolytes. We find that urea denaturation governed by transfer free energy differences is dominated by vdW attractions, whereas TMAO exerts its effect by causing unfavorable electrostatic interactions both in the binary solution and mixed osmolyte solution. Analysis of the results showed no evidence in the ternary solution of disruption of the correlations among the peptide and osmolytes, nor of significant changes in the strength of the water hydrogen bond network.

Backbone additivity in the transfer model of protein solvation.

The transfer model implying additivity of the peptide backbone free energy of transfer is computationally tested. Molecular dynamics simulations are used to determine the extent of change in transfer free energy (DeltaG(tr)) with increase in chain length of oligoglycine with capped end groups. Solvation free energies of oligoglycine models of varying lengths in pure water and in the osmolyte solutions, 2M urea and 2M trimethylamine N-oxide (TMAO), were calculated from simulations of all atom models, and DeltaG(tr) values for peptide backbone transfer from water to the osmolyte solutions were determined. The results show that the transfer free energies change linearly with increasing chain length, demonstrating the principle of additivity, and provide values in reasonable agreement with experiment. The peptide backbone transfer free energy contributions arise from van der Waals interactions in the case of transfer to urea, but from electrostatics on transfer to TMAO solution. The simulations used here allow for the calculation of the solvation and transfer free energy of longer oligoglycine models to be evaluated than is currently possible through experiment. The peptide backbone unit computed transfer free energy of -54 cal/mol/M compares quite favorably with -43 cal/mol/M determined experimentally.

Trimethylamine N-oxide influence on the backbone of proteins: an oligoglycine model.

The study of organic osmolytes has been pivotal in demonstrating the role of solvent effects on the protein backbone in the folding process. Although a thermodynamic description of the interactions between the protein backbone and osmolyte has been well defined, the structural analysis of the effect of osmolyte on the protein backbone has been incomplete. Therefore, we have performed simulations of a peptide backbone model, glycine(15), in protecting osmolyte trimethylamine N-oxide (TMAO) solution, in order to determine the effect of the solution structure on the conformation of the peptide backbone. We show that the models chosen show that the ensemble of backbone structures shifts toward a more collapsed state in TMAO solution as compared with pure water solution. The collapse is consistent with preferential exclusion of the osmolyte caused by unfavorable interactions between osmolyte and peptide backbone. The exclusion is caused by strong triplet correlations of osmolyte, water, and peptide backbone. This provides a clear mechanism showing that even a modest concentration of TMAO forces the protein backbone to adopt a more collapsed structure in the absence of side chain effects.

Osmolyte solutions and protein folding.

In this brief review we discuss the evolution of recent thought regarding the role and mechanism of osmolytes with respect to protein stability. Osmolytes are naturally occurring intracellular compounds that change the protein folding landscape. Contributions from experiments are considered in the context of current theory and simulation results.

Optimized acellular nerve graft is immunologically tolerated and supports regeneration.

To replace the autologous graft as a clinical treatment of peripheral nerve injuries we developed an optimized acellular (OA) nerve graft that retains the extracellular structure of peripheral nerve tissue via an improved chemical decellularization treatment. The process removes cellular membranes from tissue, thus eliminating the antigens responsible for allograft rejection. In the present study, the immunogenicity and regenerative capacity of the OA grafts were tested. Histological examination of the levels of CD(8+) cells and macrophages that infiltrated the OA grafts suggested that the decellularization process averted cell-mediated rejection of the grafts. In a subsequent experiment, regeneration in OA grafts was compared with that in isografts (comparable to the clinical autograft) and two published acellular graft models. After 84 days, the axon density at the midpoints of OA grafts was statistically indistinguishable from that in isografts, 910% higher than in the thermally decellularized model described by Gulati (J. Neurosurg. 68, 117, 1988), and 401% higher than in the chemically decellularized model described by Sondell et al. (Brain Res. 795, 44, 1998). In summary, the results imply that OA grafts are immunologically tolerated and that the removal of cellular material and preservation of the matrix are beneficial for promoting regeneration through an acellular nerve graft.