Protein stabilization by compatible solutes

Abstract

Heteronuclear NMR relaxation measurements and hydrogen exchange data have been used to characterize protein dynamics in the presence or absence of stabilizing solutes from hyperthermophiles. Rubredoxin from Desulfovibrio gigas was selected as a model protein and the effect of diglycerol phosphate on its dynamic behaviour was studied. The presence of 100 mM diglycerol phosphate induces a fourfold increase in the half-life for thermal denaturation of D. gigas rubredoxin [Lamosa, P., Burke, A., Peist, R., Huber, R., Liu, M.Y., Silva, G., Rodrigues-Pousada, C., LeGall, J., Maycock, C. & Santos, H. (2000) Appl. Environ. Microbiol. 66, 1974Microbiol. 66, -1979. A model-free analysis of the protein backbone relaxation parameters shows an average increase of generalized order parameters of 0.015 reflecting a small overall reduction in mobility of fast-scale motions. Hydrogen exchange data acquired over a temperature span of 20°C yielded thermodynamic parameters for the structural opening reactions that allow for the exchange. This shows that the closed form of the protein is stabilized by an additional 1.6 kJAEmol )1 in the presence of the solute. The results seem to indicate that the stabilizing effect is due mainly to a reduction in mobility of the slower, larger-scale motions within the protein structure with an associated increase in the enthalpy of interactions.Keywords: chemical exchange; compatible solutes; protein dynamics; rubredoxin; thermostability.Protein stability, activity and dynamics are interrelated issues with great importance not only in physiological processes but also in protein engineering. The evolution of protein structures towards extreme thermostability was vital for hyperthermophiles, microorganisms thriving near the boiling point of water. In general, the proteins of these organisms are intrinsically resistant to heat denaturation. However, hyperthermophiles also possess intracellular proteins that are not particularly stable, implying the existence of alternative strategies for their stabilization in vivo [1,2]. Hyperthermophiles accumulate high levels of charged organic osmolytes in response to supra-optimal growth temperatures, and this observation led to the hypothesis that these compounds play a role in thermoprotection of macromolecules in vivo [3,4]. This view is supported by in vitro studies showing that these osmolytes protect proteins against heat [1,[5][6][7][8]. Nevertheless, the molecular basis for this well established stabilization phenomenon remains elusive.Several possible mechanisms for protein stabilization by osmolytes have been proposed [9][10][11]. Arakawa and Timasheff [12,13] proposed a preferential hydration model to explain protein stabilization by compatible solutes: solute molecules are excluded from the protein surface, thereby making denaturation entropically less favourable. In conformity, exclusion factors have been measured for a variety of organic solutes and salts [14][15][16], however, the correlation between exclusion factors and the degree of protection ...

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