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 denaturated 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.