Protein aggregation appears to originate from partially unfolded conformations that are sampled through stochastic fluctuations of the native protein. It has been a challenge to characterize these fluctuations, under native like conditions. Here, the conformational dynamics of the full-length (23-231) mouse prion protein were studied under native conditions, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS). The slowest fluctuations could be associated with the folding of the unfolded state to an intermediate state, by the use of microsecond mixing experiments. The two faster fluctuations observed by PET-FCS, could be attributed to fluctuations within the native state ensemble. The addition of salt, which is known to initiate the aggregation of the protein, resulted in an enhancement in the time scale of fluctuations in the core of the protein. The results indicate the importance of native state dynamics in initiating the aggregation of proteins.

No abstract

Defining the role of non-native interactions in directing the course of protein folding or unfolding reactions has been a difficult challenge. In particular, the extent to which such interactions play a productive role by stabilizing the structures of transition states (TSs) found on the folding and unfolding pathways of proteins is not known. On the contrary, it is thought that the TSs are expanded forms of the N state stabilized by native interactions, and it is not known whether non-native interactions can modulate TS structure. In this study of the unfolding of the SH3 domain of PI3 kinase using a microsecond mixing methodology, partial non-native structure formation is shown to occur initially during unfolding. The TS of this partial "folding during unfolding" reaction is more compact than the N state: the apparent rate constant of Trp53 burial during this reaction decreases with an increase in denaturant concentration. Kinetic studies of the unfolding of mutant variants suggest that the unusually compact TS is stabilized by interactions not present in N and that these non-native interactions are hydrophobic in nature. It was determined that mutation could be used to tune the degree of compaction in the TS.