The Structural and Energetic Bases of Extreme Thermal Stability in Proteins

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96-04213 Dahlquist This study is designed to elucidate the nature of hyperthermostability in the CheY protein from Thermotoga maritima (TMY), which undergoes reversible thermal unfolding at 95oC. The CheY isolated from Bacillus subtilis is the same length and is 75% identical in amino acid sequence to TMY but denatures about 35oC lower in temperature. Each of the 30 residues that differ in the two proteins will be substituted in order to monitor the contribution that each makes to the overall stability of the protein. Selected mutants will be examined by a variety of physical methods including circular dichroism, solution nuclear magnetic resonance methods, differential scanning calorimetry and X- ray crystallography for possible relationships between the thermal stability of a protein and its structural and dynamic properties. A thermally-induced structural change in TMY that occurs about 50o below its thermal unfolding will also be characterized. This transition, similar to those observed in other proteins from hyperthermophilic sources, results in extensive line broadening and chemical shift changes in the 15N-1H correlation spectra of TMY. The information so gained in this study should provide a rich experimental database for the understanding of the structural and energetic contribution to the exceptional thermal stability seen in proteins from hyperthermophilic sources. Proteins from organisms that live at very high temperatures (hyperthermophiles) are much more stable than similar proteins from organisms that live near body temperature (mesophiles). This study undertakes a systematic investigation of the source of stability in a small, well behaved protein, CheY, which is involved in sensory signaling in both hyperthermophiles and mesophiles. About 20 amino acids of this protein, isolated from a hyperthermophile has, are likely to account for a near 50oC increase in its thermal stability, as compared to the similar protein from a mesophilic source. These residues will be varied by sitedirected mutation so that the role of these residues in the stability of the protein and its structure, as determined by X-ray diffraction and nuclear magnetic resonance methods, can be recognized. In addition, there is a change in structure as temperature increases that occurs near body temperature in the CheY protein from the hyperthermophile. This structural change may reflect a change from a catalytically inactive form at low temperature to a functional form at high temperature. This change and the reasons that it occurs will be characterized to yield a general understanding of how proteins function under extreme environmental conditions.