Protein stabilization by osmolytes from hyperthermophiles - Effect of mannosylglycerate on the thermal unfolding of recombinant nuclease a from Staphylococcus aureus studied by picosecond time-resolved fluorescence and calorimetry

• Type: Article in International Scientific Journal
• Date: 2004

Authors

Faria, TQ (Author) Other; Lima, JC (Author) Other; Bastos, M (Author) FCUP; Macanita, AL (Author) Other; Santos, H (Author) Other

Journal

Title: Journal of Biological Chemistry

Vol. 279

Pages: 352-352

ISSN: 0021-9258

Publisher: Elsevier

Indexing

ISI Web of Knowledge - 55 Citations

Scopus - 58 Citations

Scientific classification

FOS: Natural sciences > Biological sciences

Other information

Authenticus ID: P-000-2JA

DOI: 10.1074/jbc.m408806200

Abstract (EN): 2-O-alpha-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 degreesC 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 degreesC 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.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 12