Equilibrium and pressure-jump relaxation studies of the conformational transitions of P13MTCP1

The conformational transitions of a small oncogene product, P13(MTCP1), have been studied by high-pressure fluorescence of the intrinsic tryptophan emission and high-pressure 1D and 2D. H-1-N-15 NMR. While the unfolding transition monitored by fluorescence is cooperative, two kinds of NMR spectral changes were observed, depending on the pressure range. Below similar to200 MPa, pressure caused continuous, non-linear shifts of many of the N-15 and H-1 signals, suggesting the presence of an alternate folded conformer(s) in rapid equilibrium (tau much less than ms) with the basic native structure. Above similar to200 MPa, pressure caused a sharp decrease in the intensity of the folded proteins signals, while the peaks corresponding to disordered structures increased, yielding a free energy of unfolding change of 6.0 kcal/mol and associated volume change of -100 ml/mol, in agreement with the fluorescence result. Differential scanning calorimetry also reveals two transitions between 21 and 65 degreesC, confirming the existence of an additional species under mildly denaturing conditions. We report here a real-time observation of pressure-jump unfolding kinetics by 2D NMR spectroscopy on P13(MTCP1) made possible due to its very long relaxation times at high pressure revealed by fluorescence studies. Within the dead-time after the pressure-jump, the NMR spectra of the native conformer changed to those of the transient conformational species, identified in the equilibrium studies, demonstrating the equivalence between a transient species and an equilibrium excited state. After these rapid spectral changes, the intensities of all of the individual N-15-H-1 cross-peaks decreased gradually, and those of the disordered structure increased, consistent with the slow relaxation to the unfolded form at this pressure. Rate constants of unfolding monitored at individual amide sites within the P-barrel were similar to those obtained from fluorescence and from side-chain protons in the hydrophobic core region, consistent with nearly cooperative unfolding. However, some heterogeneity in the apparent unfolding rate constants is apparent across the sequence and can be understood as non-uniform effects of pressure on the unfolding rate constant due to non-uniform hydration. (C) 2002 Elsevier Science Ltd. All rights reserved.