Application of dynamic light scattering to studies of protein folding kinetics

The applicability of dynamic light scattering to studies of the kinetics of unfolding and refolding reactions of proteins is discussed and demonstrated experimentally. The experimental set-up and the data acquisition and data evaluation schemes that have been optimized for kinetic experiments are described. The relationship of the signal-to-noise ratio to the minimum data acquisition time that is needed to obtain results of sufficiently high precision is discussed. It turns out that the attainable time resolution is of the order of a few seconds for proteins with molar masses of about 50,000 g · mol^–1 and concentrations of 1 g · l^–1. Thus, DLS is too slow to follow conformational changes in the subsecond region, but it is useful for studies of unfolding-refolding reactions of proteins that proceed with time constants in the range of seconds or minutes. This is demonstrated by investigations of the kinetics of the cold denaturation of 3-phosphoglycerate kinase from yeast.Abbreviations DLS dynamic light scattering - PGK 3-phosphoglycerate kinase - EDTA ethylenediamine tetraacetic acid - GuHCl guanidine hydrochloride - DTT dithiothreitol