METHOD DEVELOPMENT AND RESULTS OF PROTEIN THERMAL-DENATURATION ANALYSIS BY H-1-NMR

NMR spectroscopy is a powerful technique for probing protein structure , stability, function and folding. Any expansion of NMR to other field s, such as engineering, could only be a benefit. Since protein thermal stability studies can provide useful information for protein processi ng in the biopharmaceutical industry, the applicability of one-dimensi onal proton (1D H-1) NMR for thermal denaturation midpoint (T-m) and l ongitudinal relaxation times (T-1) for thermal stability data were inv estigated for the globular protein lysozyme. The considerations for pr otein NMR spectral analysis through temperature changes are elaborated upon. These considerations may also be of great value for protein, pe ptide, carbohydrate and oligonucleotide spectral analysis through chan ges other than temperature, such as pressure, pH, concentration and so lute interactions. Since the experimental considerations elaborated up on were used for both T-1 and T-m analysis, they should also apply to the determination of other NMR-derived data. The denatured and native H15 C2H hen lysozyme protons were used to estimate T-m to within 2.3%. The native H15 C2H H-1 T-1 is greater than the denatured H-1 T-1 at a ll temperatures ascertained in the presence or absence of polyethylene glycol 1000 (PEG 1000). The difference in the native and denatured H1 5 C2H T-1 values indicates that the surface-located histidine residue experiences a differing local native conformation than the denatured l ocal averaged conformation state. The H15 C2H denatured H-1 T-1 is sho rter in 20% PEG 1000 than at 0% PEG 1000. The lower denatured T-1 in t he presence of PEG indicates that PEG may interact with the denatured protein. (C) 1997 John Wiley & Sons, Ltd.