A solid-state NMR study of protein hydration and stability

Purpose. The mobility of protein in powders at different hydration levels w as studied in relation to aggregation and activity. Methods. Magic angle spinning C-13, N-15, H-1, H-2, and O-17 NMR techniques were used to determine changes in the mobility of surface residues in prot eins as a function of hydration and related to changes in activity. NMR rel axation measurements of high frequency (omega(0) Ti) and low frequency (ome ga(1), T-1 rho) motions have been carried out on lyophilized DNase, insulin and lysozyme stored at different relative humidities. Moisture-induced agg regation and enzymatic activity of the lyophilized proteins was determined by high performance size exclusion chromatography and bioassays. Results. There was little change in T-1 rho observed with increasing humidi ty. The results show, however, that there is a decrease in T-1 for DNase, i nsulin and lysozyme at relative humidities ranging from 0-98%, and we propo se that the reduction in T-1 is related to the aggregation susceptibility o f proteins during storage at different humidities. The water mobility was d etermined directly using O-17 NMR experiments. We found that as the amount of weakly-bound water increases, the protein surface mobility decreases and is coupled with increased aggregation. Aggregation measurements at differe nt humidities were correlated with bioassays for lysozyme and found to be c onsistent with the hydration data. Conclusions. Mobility of protein molecules was determined by solid-state NM R over a wide range of % RH and it was found that water content leads to a change in mobility of protein molecules. The aggregation and activity of pr oteins were strongly correlated to change in molecular mobility.