Nondestructive determination of protein structural stability

The so called protein dynamical transition has long been associated with thermally activated excitations of the solvent adjacent to the polypeptide surface. Recently we reported the somewhat surprising result that the low temperature (<270K) terahertz absorption measurements of a series of red fluorescent protein showed an increase in the dynamical transition temperature with photobleaching, and that this increase correlated with an increase in the melting temperatures. Here we show that the frequency dependence of the absorption sharply changes with photobleaching, and this frequency dependence can be decomposed into a water vibrational part and protein vibrational part. While the temperature dependence of the water component remains unchanged with photobleaching, the protein component has a dramatic change in the temperature dependence, revealing that A) the THz absorbance can measure the protein dynamics in solution phase and B) the temperature dependence of the picosecond dynamics reflect the overall protein structural stability.