Protein Stability in Ice

Abstract

This study presents an experimental approach, based on the change of Trp fluorescence between native and denatured states of proteins, which permits to monitor unfolding equilibria and the thermodynamic stability (DeltaG degrees ) of these macromolecules in frozen aqueous solutions. The results obtained by guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, in the temperature range from -8 to -16 degrees C, demonstrate that the stability of the native fold may be significantly perturbed in ice depending mainly on the size of the liquid water pool (V(L)) in equilibrium with the solid phase. The data establish a threshold, around V(L)=1.5%, below which in ice DeltaG degrees decreases progressively relative to liquid state, up to 3 kcal/mole for V(L)=0.285%. The sharp dependence of DeltaG degrees on V(L) is consistent with a mechanism based on adsorption of the protein to the ice surface. The reduction in DeltaG degrees is accompanied by a corresponding decrease in m-value indicating that protein-ice interactions increase the solvent accessible surface area of the native fold or reduce that of the denatured state, or both. The method opens the possibility for examining in a more quantitative fashion the influence of various experimental conditions on the ice perturbation and in particular to test the effectiveness of numerous additives used in formulations to preserve labile pharmaco proteins.

Protein aggregation: Pathways, induction factors and analysis

“…Freezing introduces complex physical and chemical changes including creation of new ice/solution interfaces, [48][49][50] adsorption to container surfaces, 50 cryoconcentration of the protein and solutes, 51,52 and pH changes due to crystallization of buffer components. 53 These effects are regarded as possible causes of freezing-induced protein denaturation and aggregation.…”

Effect of Lyoprotectant on the Solubility and Structure of Silk Sericin

“…Removing solvent, mostly water, is important because a liquid state protein or peptide is unstable to store. It is due to the hydrolysis or oxidation of protein in the presence of water, which results in various kinds of denaturation such as gelation, aggregation and degradation (Kreilgaard et al, 1998;Gabellieri et al, 2003;Gabellieri et al, 2006;Strambini et al, 2007). However, the removal of water may cause structural changes in the secondary and/or tertiary structure of protein.…”

Heterogeneity of Protein Environments in Frozen Solutions and in the Dried State

“…As evidence to the contrary, i.e., of interaction of non-antifreeze-proteins with ice interface, studies reported in [38,39] are commonly invoked, and discussed below in some detail. Conformational changes of globular proteins were studied by employing the phosphorescence emission of tryptophan (Trp) residues as a monitor of the conformational changes of six proteins in response to variations in conditions of the medium [38].…”