Temperature-jump-induced refolding of ribonuclease A: a time-resolved FTIR spectroscopic study

FTIR difference spectroscopy has been used for the first time to investigate the kinetics of secondary structure formation during refolding. The refolding process of ribonuclease A (RNase A) as a model system was induced by applying a temperature-jump of 60 degrees. The temperature-jump was triggered by rapidly injecting a small volume of the thermally unfolded protein solution at 80 degrees C into a special cuvette system kept at 20 degrees C. The dead-time of the injection and the time resolution of the FTIR spectrometer permitted the observation of refolding processes in a time window ranging from 170 ms to several minutes. Specifically, the formation of beta-structures and the disappearance of irregular conformations could be observed in this time interval.