Mutational and structural-based analyses of the osmolyte effect on protein stability

Area of Science:

• Biochemistry
• Protein Science
• Molecular Biology

Background:

• Osmolytes are natural compounds that enhance protein conformational stability.
• The osmophobic theory suggests osmolytes interact unfavorably with the protein backbone, driving stability.
• Previous studies used amino acid transfer Gibbs energy (Deltag) to support this theory.

Purpose of the Study:

• To investigate the effect of sarcosine on the conformational stability of RNase Sa and its mutants.
• To validate the osmophobic theory using experimental and computational approaches.
• To assess the utility of molecular dynamics (MD) simulations in modeling denatured protein structures.

Main Methods:

• Thermal denaturation analysis of RNase Sa and mutants in the presence of sarcosine.
• Nonlinear least-squares analysis to fit denaturation curves to a two-state model.
• Mutational analysis and calculation of transfer Gibbs energy (Deltag) for osmolytes.
• Structural analysis using molecular dynamics (MD) simulations of model denatured protein structures.

Main Results:

• Sarcosine significantly increased the conformational stability (DeltaG) of RNase Sa proteins.
• Experimental and calculated changes in osmolyte effect upon mutation (Delta(m)Delta(o)DeltaG) showed good agreement.
• Two-part, unfolded models of denatured structures, simulated via MD, yielded calculated Delta(o)DeltaG values consistent with experimental data.
• The osmophobic theory effectively explains the observed osmolyte effect on protein stability.

Conclusions:

• Sarcosine acts as a stabilizing osmolyte for RNase Sa, consistent with the osmophobic theory.
• MD simulations provide a valuable tool for predicting denatured protein structures and understanding osmolyte interactions.
• The findings reinforce the applicability of the osmophobic theory in explaining osmolyte-induced protein stabilization.