Abstract
The temperature dependence of the overall dimensions of the denatured state ensemble (DSE) of proteins remains unclear. Some studies indicate compaction of the DSE at high temperatures, whereas others argue that dimensions do not decrease. The degree of compaction or expansion in the cold-denatured state has been less studied. To investigate the temperature dependence of unfolded state dimensions, small angle x-ray scattering measurements were performed in native buffer in the absence of denaturant for a designed point mutant of the C-terminal domain of L9, a small cooperatively folded α-β protein, at 14 different temperatures over the range of 5-60°C. The I98A mutation destabilizes the domain such that both the DSE and the folded state are populated at 25°C in the absence of denaturant or extreme pH. Thermal unfolding as well as cold unfolding can thus be observed in the absence of denaturant, allowing a direct comparison of these regimes for the same protein using the same technique. The temperature of maximal stability, T(s), is 30°C. There is no detectable change in R(g) of the unfolded state as the temperature is increased above T(s), but a clear expansion is detected as the temperature is decreased below T(s). The R(g) of the DSE populated in buffer was found to be 27.8 ± 1.7 Å at 5°C, 21.8 ± 1.9 Å at 30°C, and 21.7 ± 2.0 Å at 60°C. In contrast, no significant temperature dependence was observed for the value of R(g) measured in 6 M guanidine hydrochloride. The small angle x-ray scattering data reported here indicate clear differences between the cold- and thermal-unfolded states and show that there is no significant compaction at elevated temperatures.