Thermodynamic analysis of the nondenaturational conformational change of baker’s yeast phosphoglycerate kinase at 24 °C
Section snippets
Reagents
Baker’s yeast phosphoglycerate kinase (product number P7634), GuHCl, and the sodium phosphate buffer components were obtained from Sigma Chemical Company. All measurements were performed in 150 mM sodium phosphate, pH = 7.0.
Equilibrium thermodynamics data acquisition
To obtain data for the high-temperature portion of the stability curve, PGK solutions greater than 2 mg/mL and of known volume were prepared in buffer and then allowed to dialyze for at least 72 h against either 0.84, 0.95, 1.01, 1.09, 1.12 or 1.21 M solutions of GuHCl at pH = 7.0
Results
Our first objective is to obtain a stability curve—a plot of the Gibbs free energy of unfolding vs. temperature—for PGK under physiological conditions. Our methodology does not assume that the conformation which predominates at high temperature persists at low temperature; i.e., we do not simply obtain unfolding data in the high temperature regime and extrapolate into the low temperature regime. In fact, we assume there will be such a conformational change as we have observed this with bovine
Discussion
The primary purpose of this investigation is to establish that PGK undergoes a temperature-induced, nondenaturational conformational change at a temperature between the optimal thriving temperature of S. cerevisiae (which is 27.5 °C [4]) and 4 °C where crystals for the structure determination seem to be formed. This is done by the construction of a stability curve of the enzyme with care taken to obtain stability data over a wide temperature range. The existence of a conformational change at 24 °C
Acknowledgments
This work was supported by a departmental Grant from the Welch Foundation, American Chemical Society Petroleum Research Fund grant (#43241-B4), and a Grant from the State of Texas Norman Hackerman Advanced Research Program. We thank Kathy Westfall for helpful discussion.
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2020, Proceedings of the National Academy of Sciences of the United States of America