A solid-state NMR study of protein hydration and stability
A solid-state NMR study of protein hydration and stability
复制标题
DOI：
10.1023/a:1011993620177
复制
发表时间：
1998-12-01
影响因子：
3.7
通讯作者：
Chan, HK
中科院分区：
文献类型：
--
作者：
Separovic, F;Lam, YH;Chan, HK
目的。研究了不同水合水平粉末中蛋白质的流动性与聚集性和活性的关系。魔角旋转C-13， N-15, H-1， H-2和O-17核磁共振技术被用来确定蛋白质表面残基的迁移率的变化，作为水合作用的功能，并与活性的变化有关。对储存在不同相对湿度下的冻干dna酶、胰岛素和溶菌酶进行了高频（ω (0) Ti）和低频（ω (1), T-1 rho）运动的核磁共振弛豫测量。用高效粒径隔离色谱法和生物测定法测定了冻干蛋白在水分诱导下的聚集和酶活性。随湿度的增加，T-1 rho变化不大。结果表明，在0 ~ 98%的相对湿度范围内，dna酶、胰岛素和溶菌酶的T-1含量降低，这可能与不同湿度条件下蛋白质在贮藏过程中的聚集敏感性有关。用O-17核磁共振实验直接测定了水的迁移率。我们发现，随着弱结合水数量的增加，蛋白质表面迁移率降低，并伴有聚集增加。在不同湿度下的聚集测量与溶菌酶的生物测定相关联，并发现与水化数据一致。蛋白质分子的迁移率是由固态核磁共振在% RH的宽范围内确定的，发现水的含量导致蛋白质分子迁移率的变化。蛋白质的聚集和活性与分子迁移率的变化密切相关。
Purpose. The mobility of protein in powders at different hydration levels was 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 proteins as a function of hydration and related to changes in activity. NMR relaxation measurements of high frequency (omega(0) Ti) and low frequency (omega(1), T-1 rho) motions have been carried out on lyophilized DNase, insulin and lysozyme stored at different relative humidities. Moisture-induced aggregation 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 humidity. The results show, however, that there is a decrease in T-1 for DNase, insulin and lysozyme at relative humidities ranging from 0-98%, and we propose that the reduction in T-1 is related to the aggregation susceptibility of proteins during storage at different humidities. The water mobility was determined 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 different humidities were correlated with bioassays for lysozyme and found to be consistent with the hydration data.Conclusions. Mobility of protein molecules was determined by solid-state NMR 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 proteins were strongly correlated to change in molecular mobility.