Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Evidence for Initial Non-specific Polypeptide Chain Collapse during the Refolding of the SH3 Domain of PI3 Kinase
Proteins are evolutionarily selected heteropolymers, but their response to solvent change appears to be very similar to that of simple homopolymers (1, 2). The unfolded protein chains undergo global contraction when transferred from a good to a bad solvent (3, 4). This ultrafast compaction of the unfolded state of a protein (collapse) channels it to the unique native structure by reducing its conformational space. The collapse reaction of any polypeptide chain has been assumed to be driven by non-specific hydrophobic interactions (3, 5). Alternatively, the driving force of the collapse reaction could also be the formation of backbone hydrogen bonds at low concentration of denaturant (6, 7). For some recent provocative discussion on protein folding, see Mittal et al. (8), Matthews (9) Scheraga (10) and others published in the February 2011 issue of this Journal.
In the present study, the refolding of the PI3K SH3 from the guanidine hydrochloride (GdnHCl)-unfolded state was probed with millisecond (stopped flow) and sub-millisecond (continuous flow) measurements of the change in tyrosine fluorescence, circular dichroism, ANS fluorescence and three-site fluorescence resonance energy transfer (FRET) efficiency (11). Previously, the refolding of this protein appeared two-state (12). Presently, our studies show that the folding of the protein commences via the rapid (complete within 150 μs) formation of a collapsed ensemble in a transition that is gradual and without any significant accumulation of secondary structure. All three intra-molecular distances collapsed to the same extent indicating that the compaction was synchronous as that expected for a coil to globule transition of a simple homopolymer as a consequence of solvent change (13). These results highlight the homopolymer nature of the unfolded polypeptide chain of the PI3K SH3. Furthermore to investigate the role of intra-chain hydrogen bonding in the collapse reaction, the folding was initiated by dilution of the urea-unfolded state (11). The extent of compaction seen for one of the intra-molecular distance was similar to that observed for the GdnHCl induced unfolded state.
To elucidate the importance of a non-specific collapsed ensemble in the subsequent structure formation of the PI3K SH3, an attempt was made to tune folding conditions such that a specific structured component of the collapsed ensemble could be preferentially populated. With the above objective, the effect of 500 mM sodium sulphate on the refolding of the PI3K SH3 was studied using multiple spectroscopic probes. Results indicate the formation of a specifically collapsed intermediate that has greater ANS binding than the previously reported non-specific ensemble (8). Two intra-molecular distances in this collapsed ensemble show greater contraction than just a solvent induced compaction, indicating the formation of a specific structured ensemble, before the rate limiting step of folding. Interestingly, one of the FRET pairs indicates the formation of native-like distance in the first millisecond of the refolding reaction.
Abbreviations used: FRET, fluorescence resonance energy transfer; PI3K SH3, SH3 domain of PI3 kinase; ANS, 1-anilino-naphthalene-8-sulfonate.