Factor Defining the Effects of Glycine Betaine on the Thermodynamic Stability and Internal Dynamics of Horse Cytochrome c

Abstract

A compatible osmolyte such as glycine betaine (GB) and low concentrations of a denaturant constrain the internal dynamics of natively folded carbonmonoxycytochrome c (NCO) at pH 7.0. GB and subdenaturing concentrations of guanidine hydrochloride (GdnHCl) or urea have a cumulative effect on the constrained dynamics of NCO. At higher denaturant concentrations, large-scale unfolding fluctuations dominate the dynamics and inclusion of GB opposes the structural fluctuations that cause unfolding of the protein. These deductions are made from kinetic and thermodynamic parameters measured for the CO dissociation reaction of NCO at varying concentrations of denaturant in the absence and presence of 1.0 M GB. Intermolecular docking between horse ferrocytochrome c and a denaturant or GB reveals that the denaturant-mediated constrained dynamics of the protein is due to polyfunctional interactions between the denaturant and different groups of protein while the GB-mediated restricted dynamics of the protein arises from both the direct interactions of GB with different side chains of Lys or Arg residues of the protein and indirect interactions of GB with the protein surface. Thermodynamic analysis of the thermal and GdnHCl-induced unfolding curves of ferrocytochrome c measured in the absence and presence of 1.0 M GB at pH 7.0 indicates that GB increases the thermodynamic stability of ferrocytochrome c at neutral pH. Analysis of thermal and urea-induced unfolding curves of ferricytochrome c measured at different GdnHCl concentrations in the absence and presence of 0.5-1.0 M GB at pH 7.0 and 3.8 suggests that GB counteracts the destabilizing effect of the denaturant at pH 7.0 but exhibits an additive effect on the destabilizing effect of the denaturant at pH 3.8.

Coherent Experimental and Simulation Approach To Explore the Underlying Mechanism of Denaturation of Stem Bromelain in Osmolytes

“…64,65 Furthermore, due to charge−π interactions, betaine favorably interacts with the aromatic residues (Trp and Phe) of BM. 9,26 Besides, BM possesses net positive charge on its surface at physiological pH (pI of BM > 7), thereby, more betaine molecules may favorably bind to the surface of the protein through direct interactions with the Lys, Arg, His, or the N−H group of the peptide bond as well as through some indirect interactions. 9,26 With increase in the concentration of betaine, there may be no more water molecules available to hydrate betaine molecules and less water is there for competition with betaine for the protein surface.…”

Protein folding and stability in the presence of osmolytes

“…It should be noted that osmolytes do not cause significant changes in the structures of native proteins [57,82,83]. However, osmolytes are capable of influencing the dynamics of structural changes of native proteins by reducing the heterogeneity of the distribution of the protein conformers in solution [84][85][86][87][88]. In turn, the introduction of osmolytes into solutions that contain denatured or partially denatured proteins results in the compaction of protein structure and decreases the diversity of its conformations [65,[89][90][91][92].…”

Biomolecular Crowding Arising from Small Molecules, Molecular Constraints, Surface Packing, and Nano-Confinement

“…This essentially suggests that trehalose protects the folded state from unfolding via the excluded volume effect. Additionally, a recent study by Jain et al 16 hypothesized that the ability of the osmolyte glycine betaine (GB) to counteract the destabilizing effect of chemical denaturants and temperature changes on ferricytochrome c and ferrocytochrome c is partially the result of zwitterionic GB being excluded from the protein surface in much the same way as TMAO. Moreover, using MD simulations, Zhou and coworkers 17 observed that in the presence of guanidinium chloride (GdmCl), urea actually induced a crowding effect on two proteins, hen egg-white lysozyme and protein L, causing them to collapse.…”