Water-protein interactions in the molten-globule state of carbonic anhydrase b: an NMR spin-diffusion study

We have used the homonuclear Overhauser effect (NOE) to characterize a model protein: carbonic anhydrase B. We have obtained NOE difference spectra for this protein, centering the on-resonance signals either at the methyl-proton or at the water-proton signals. The spin-diffusion spectra obtained as a function of protein concentration and temperature provide direct evidence of much greater protein-water interaction in the molten-globule state than in the native and denatured states. Furthermore, although the protein loses its gross tertiary structure in both the molten-globule and denatured states, it remains almost as compact in its molten-globule state as it is in the native state. The spin-diffusion spectra, obtained as a function of a variable delay time after the saturation pulse, allowed us to measure the relaxation times of several types of proton in the solution. These spectra contain enough information to distinguish between those water molecules solvating the protein and the free ones present as bulk water.