From Model Compounds to Proteins: A Volumetric Study of the Stabilizing Action of Glycine Betaine

Abstract

Glycine betaine is a strong protecting osmolyte capable of stabilizing the folded conformations of proteins. A lingering problem has been to determine the molecular mechanisms underlying the enhancement of protein stability. One approach to tackle this problem is to examine the differential solvation properties of proteins and their components in water and glycine betaine. We present a new statistical thermodynamic formalism to quantify the individual binding affinities of glycine betaine for the polar and nonpolar moieties of globular proteins. This approach enables one to rationalize partial molar volume, V°, and adiabatic compressibility, Ks°, measurements in terms of interactions of glycine betaine with polar and nonpolar protein groups. We characterize in this way glycine betaine interactions with ribonuclease A, lysozyme, cytochrome C, and ovalbumin. To this end, we use our previous volumetric data on N-acetyl amino acid amides (models for amino acid side chains) and oligoglycines (models for peptide backbone) in solutions of glycine betaine at concentrations ranging from 0 to 4 M. We determine the average binding affinity, k, of glycine betaine for the proteins as well as changes in the volume, ΔVo, and adiabatic compressibility, ΔKso, associated with the binding of a glycine betaine molecule to the proteins. We find good agreement between these characteristics determined for proteins and their low molecular weight model compounds. These results suggest little or no cooperativity for glycine betaine interactions with proteins. Our experimental results in conjunction with the statistical thermodynamic formalism that we developed offer a new paradigm for investigating weak protein-ligand interactions.