Origin of apparent fast and non-exponential kinetics of lysozyme folding measured in pulsed hydrogen exchange experiments.

Folding of lysozyme at pH 5.2 is a complex processes. After rapid collapse (<1 ms) kinetic partitioning into a slow and fast folding pathway occurs. The fast pathway leads directly to the native structure (N), whereas the slow pathway goes through a partially folded intermediate (I(1)) with native-like secondary structure in the alpha-domain. This mechanism is in agreement with data from a large number of spectroscopic probes, from changes in the radius of gyration and from measurements on the time-course of the populations of the different species. Results from pulsed hydrogen exchange experiments, in contrast, revealed that the secondary structure of I(1) and of N is formed significantly faster than changes in spectroscopic properties occur and showed large variations in the protection kinetics of individual amide sites. We investigated the molecular origin of the rapid amide protection by quantitatively simulating all kinetic processes during the pulse-labeling experiments. Absorbance and fluorescence-detected folding kinetics showed that the early events in lysozyme folding are accelerated under exchange conditions (pH 9.2) and that a change in folding mechanism occurs due to the transient population of an additional intermediate (I(2)). This leads to kinetic competition between exchange and folding during the exchange pulse and to incomplete labeling of amide sites with slow intrinsic exchange rates. As a result, apparently faster and non-exponential kinetics of amide protection are measured in the labeling experiments. Our results further suggest that collapsed lysozyme (C) and I(1) have five and ten-times reduced free exchange rates, respectively, due to limited solvent accessibility.