High-resolution NMR study of the pressure-induced unfolding of lysozyme.
The pressure-induced reversible unfolding of lysozyme was investigated by high-resolution proton magnetic resonance spectroscopy by following the proton spectra of the following residues: His-15 epsilon 1, Trp-28 epsilon 3, Leu-17 delta 2, Cys-64 alpha, and Trp-108 epsilon 3. The experiments were performed at pH 3.9 and 68.5 degrees C in the pressure range from 1 bar to 5 kbar both in the absence and presence of tri-N-acetylglucosamine (tri-NAG). From the pressure-induced changes of the equilibrium between the native and denatured forms of lysozyme, the reaction volumes (delta V) were calculated for each residue. Small but statistically significant differences in delta V were found for residues located in different regions of the protein. For example, delta V for the disulfide bonded Cys-64 alpha is smaller than the delta V's found for the other residues. In particular, the effect of tri-NAG binding to lysozyme was a change of delta V from -10.3 +/- 0.6 cm3/mol to -18.1 +/- 1.7 cm3/mol for the Trp-108 epsilon 3 residue which is located close to the active site. It is important to note that the Cys-64 alpha residue also senses the binding of the substrate analog. The ability to detect statistically significant differences for delta V of individual residues located in different regions of lysozyme represents the main result of these experiments.