Towards Understanding Hyperthermostability of Rubredoxin from Pyrococcus furiosus

Author: Cavagnero, Silvia

Year: 1997

Degree: Dissertation (Ph.D.)

Advisor: Chan, Sunney I.

Committee Members: Rees, Douglas C.; Roberts, John D.; Richards, John H.; Mayo, Stephen L.; Chan, Sunney I.

Option: Chemistry

DOI: 10.7907/zehb-bx62

Abstract

Hyperthermophiles are phylogenetically primitive organisms, whose ability to thrive at temperatures up to about 110°C is unmatched by other life forms. This study focuses on the small iron protein rubredoxin (RdPf) from the hyperthermophile Pyrococcus furiosus. Understanding the physical bases for the extraordinary in vivo and in vitro properties of this model protein is complicated by the fact that its structural characteristics are extremely similar to those of its mesophilic counterparts. The first part of our work has been concerned with the controversial role of ion pairs in the thermostabilization. RdPf undergoes minimal structural perturbations upon disruption of its salt bridges. We identified an ion pair depleted RdPf "soft" form, which is able to reversibly bind the hydrophobic probe anilinonaphthalene-8-sulfonate (ANS) with micromolar affinity and 1:1 stoichiometry, while maintaining all of its other structural properties. The location of the putative binding site has been identified by electrostatic surface potential calculations. The retained low-pH structural features, coupled with an observed decrease in apparent melting temperature by about 40°C upon ion pair disruption, suggest that pH dependent intramolecular forces appreciably modulate RdPf hyperthermostability, but there must be other noncovalent forces involved in causing it.

Our next investigations compared unfolding rates and activation parameters of the hyperthermostable RdPf with those of the mesophilic rubredoxin from Clostridium pasteurianum (RdCp), and of other typical mesophilic proteins. Arrhenius plots unequivocally show that, under all experimentally accessible temperatures, the unfolding of RdPf is several orders of magnitude slower than that of all of the examined mesophilic proteins. Furthermore, a comparative study of the pH dependence of An-henius plots of RdPf and RdCp suggests a quantifiable role of ion pairs in slowing down the unfolding kinetics. This novel kinetic, as opposed to thermodynamic, role of ion pairs may be of key importance in determining folding/unfolding kinetic barriers in proteins. Another intriguing implication of our data is that, in the specific case of RdPf, projected unfolding rates at l00°C and pH 7 yield an expected lifetime of thousands of years, vs only submilliseconds to minutes for all examined mesophilic proteins. This suggests that the hyperthermostability of RdPf may be ascribed to its being kinetically trapped at the physiological temperature of Pyrococcus furiosus, rather than to traditional thermodynamic factors. However, our kinetic data should be complemented by thermodynamic studies, in order to put our hypothesis on more solid ground.

We also asked whether hyperthermostability has any bearing on the features of the energy landscapes of RdPf, which ultimately dictate its folding and unfolding mechanism. This was achieved by studying the temperature dependence of the unfolding kinetics of RdPf by a variety of complementary spectroscopic techniques. Our data show that at least three kinetic intermediates are present along the unfolding pathways. The transition state takes place late during unfolding, which suggests that it occurs early in folding. The implications of these results for the protein folding problem have been discussed.

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