Protein Stabilization by Osmolytes from Hyperthermophiles

Abstract

2-O-␣-Mannosylglycerate, a negatively charged osmolyte widely distributed among (hyper)thermophilic microorganisms, is known to provide notable protection to proteins against thermal denaturation. To study the mechanism responsible for protein stabilization, picosecond time-resolved fluorescence spectroscopy was used to characterize the thermal unfolding of a model protein, Staphylococcus aureus recombinant nuclease A (SNase), in the presence or absence of mannosylglycerate. The fluorescence decay times are signatures of the protein state, and the pre-exponential coefficients are used to evaluate the molar fractions of the folded and unfolded states. Hence, direct determination of equilibrium constants of unfolding from molar fractions was carried out. Van't Hoff plots of the equilibrium constants provided reliable thermodynamic data for SNase unfolding. Differential scanning calorimetry was used to validate this thermodynamic analysis. The presence of 0.5 M potassium mannosylglycerate caused an increase of 7°C in the SNase melting temperature and a 2-fold increase in the unfolding heat capacity. Despite the considerable degree of stabilization rendered by this solute, the nature and population of protein states along unfolding were not altered in the presence of mannosylglycerate, denoting that the unfolding pathway of SNase was unaffected. The stabilization of SNase by mannosylglycerate arises from decreased unfolding entropy up to 65°C and from an enthalpy increase above this temperature. In molecular terms, stabilization is interpreted as resulting from destabilization of the denatured state caused by preferential exclusion of the solute from the protein hydration shell upon unfolding, and stabilization of the native state by specific interactions. The physiological significance of charged solutes in hyperthermophiles is discussed.

Structural and functional characterization of a recombinant leucine aminopeptidase

“…In addition, IF confirms a higher stability of rLAP fd up to 85 • C when a red displacement of max was observed (Fig. 3B) which is consistent with previous IF responses in other protein studies [46][47][48]. For rLAP uf IF revealed that the folding process occurs below 40 • C, based on the blue shift of max , whereas the thermal denaturation takes place from 40 • C until 60 • C. Unlike to IF, DSC has a lower sensitivity to detect gradual molecular transitions of subtle exothermic enthalpies during the establishment of hydrogen bonds; however, it is capable to detect the single endothermic transition that comprises the rupture of all the non-covalent interactions.…”

Protein folding and stability in the presence of osmolytes

“…It should be noted that introduction of osmolytes into solution under conditions of their exclusion from the immediate vicinity of the protein increases the chemical potential of the systems, i.e., it destabilizes it [25][26][27][28]. Stabilization of proteins in solutions in the presence of osmolytes is due to the fact that the concentration of osmolyte molecules in the immediate vicinity of unfolded protein is lower than in the vicinity of a native protein [39,40]. Correspondingly, the difference in chemical potentials between the native and denatured protein states in solution in the presence of osmolytes is higher than in buffer solutions.…”

Calcium-dependent and -independent Binding of Soybean Calmodulin Isoforms to the Calmodulin Binding Domain of Tobacco MAPK Phosphatase-1

“…When the Trp side chain in the free target peptide is surrounded by water molecules, the fluorescence spectra are characterized by an emission centered near 355 nm. However, if the Trp residue becomes buried in a hydrophobic region when the peptide binds to CaM, the fluorescence emission undergoes a blue shift and typically an increase in the fluorescence intensity (32,33).…”