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J. Biol. Chem., Vol. 279, Issue 47, 48680-48691, November 19, 2004

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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^*

Tiago Q. Faria, João C. Lima¶, Margarida Bastos||, António L. Maçanita**, and Helena Santos

From the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, Apartado 127, 2780-156 Oeiras, ^||Centro de Investigação em Química da Universidade do Porto, Faculdade de Ciências, Universidade do Porto, R. Campo Alegre, 687, 4169-007 Porto, and the ^**Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais, 1049-001, Lisboa, Portugal

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, pico-second 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.

Received for publication, August 2, 2004 , and in revised form, August 30, 2004.

^* This work was supported in part by the European Commission, Contracts QLK3-CT-2000-00640 and COOP-CT-2003-508644, and by Fundação para a Ciência e a Tecnologia (FCT), Portugal, and FEDER, Project POCTI 35131/BIO/2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a Ph.D. grant from FCT, PRAXIS XXI/21524/99.

^¶ Present address: Rede de Química e Tecnologia/Centro de Química Fina E Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, Monte de Caparica, 2829-516 Caparica.

To whom correspondence should be addressed. Tel.: 351-21-446-9828; Fax: 351-21-442-8766; E-mail: santos{at}itqb.unl.pt.

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