Probing the native state of a two-state folding protein by high-pressure NMR spectroscopy

H distances as a function of hydrostatic pressure for the cold shock protein B from Bacillus subtilis (BsCspB), a model protein undergoing a two-state folding-to-unfolding transition within the range applicable with HP NMR spectroscopy. Two independent approaches have been pursued in this study to determine intramolecular distances: (1) making use of a reference NOE and (2) analyzing NOE build-up curves. Both approaches yield the same result: intramolecular distances existing between the sheets that define the β-barrel fold of BsCspB have been shown to respond nonuniformly to increasing hydrostatic pressure. In fact, intramolecular distances increase, decrease, or remain unchanged when hydrostatic pressure is elevated. The majority of impacted sheet-to-sheet distances analyzed in the present study increase upon increasing hydrostatic pressure, suggesting that the three-dimensional structure of native BsCspB is modified such that β strands depart from each other. This "opening" possibly facilitates the penetration of solvent molecules into the interior of BsCspB and may represent an initial step for the pressure-induced folding-to-unfolding transition.