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

The investigation of structural, dynamic and functional properties of proteins as a function of hydrostatic pressure can provide profound insights into e.g. protein folding or protein structure-function relationships. Precise structural information is required to elucidate the response a protein exhibits upon increasing hydrostatic pressure. High pressure (HP) NMR spectroscopy represents a powerful experimental approach to obtain such structural information at atomic resolution. Here, we exploit the distance dependence of the nuclear Overhauser effect (NOE) to determine intramolecular 1H - 1H 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: (i) Making use of a reference NOE and (ii) 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 elevates. 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.