Temperature induced protein unfolding and folding of RNase a studied by time-resolved infrared spectroscopy

When a protein finds its native three-dimensional structure from the unstru ctured amino-acid chain various processes spanning a large time range are r elevant. To understand the mechanism of protein folding one needs to cover the entire folding/refolding (U <-> N) reaction on a structural level. In t he case of RNase A, the main structural changes occur in the ms time range, that can be monitored with rapid-scan-FTIR spectroscopy combined with rapi d mixing techniques. To induce unfolding we inject aqueous protein solution into a hot IR cuvette and record the time course of the spectral changes. A lag phase is found when the unfolding conditions are relatively weak, sug gesting an unfolding intermediate.