Probing the nature of the blue-shifted intermediate of photoactive yellow protein in solation by NMR: Hydrogen-deuterium exchange data and pH studies

The nature of the pB intermediate of photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been probed by NMR. pi-I-dependent changes in the NMR spectrum of the dark state of PYP are shown to closely mimic ex change broadening effects observed previously in the NMR spectrum of the pB intermediate in solution. Amide H-D exchange data show that while pB retai ns a solid protected core, two regions become significantly less protected than the dark state. The amide exchange data help to rationalize why the co nformational exchange process affects the N-terminal 28-residue segment of the protein, which is not close to the site of chromophore rearrangement. A t very low pH (pH 1.7), the dark state NMR spectrum displays approximately 30 very sharp signals, which are characteristic of a portion of the molecul e becoming unfolded. Similarities between the dark state spectra at pH simi lar to3.2 and the spectra of pB suggest a model for pB in solution where th e protein exists in an equilibrium between a well-ordered state and a state in which a region is unfolded. Such a two-state model accounts for the exc hange phenomena observed in the NMR spectra of pB, and the hydrophobic expo sure and lability inferred from thermodynamic data. It is likely that in th e crystalline environment the ordered form of pB is strongly favored.