Gonnelli M, Strambini G B
Consiglio Nazionale delle Ricerche, Istituto di Biofisca, Pisa, Italy.
Biophys J. 1993 Jul;65(1):131-7. doi: 10.1016/S0006-3495(93)81069-5.
In exploring the dynamic properties of protein structure, numerous studies have focussed on the dependence of structural fluctuations on solvent viscosity, but the emerging picture is still not well defined. Exploiting the sensitivity of the phosphorescence lifetime of tryptophan to the viscosity of its environment we have used the delayed emission as an intrinsic probe of protein flexibility and investigated the effects of glycerol as a viscogenic cosolvent. The phosphorescence lifetime of alcohol dehydrogenase, alkaline phosphatase, apoazurin and RNase T1, as a function of glycerol concentration was studied at various temperatures. Flexibility data, which refer to rather rigid sites of the globular structures, point out that, for some concentration ranges glycerol, effects on the rate of structural fluctuations of alcohol dehydrogenase and RNase T1 do not obey Kramers' a power law on solvent viscosity and emphasize that cosolvent-induced structural changes can be important, even for inner cores of the macromolecule. When the data is analyzed in terms of Kramers' model, for the temperature range 0-30 degrees C one derives frictional coefficients that are relatively large (0.6-0.7) for RNase T1, where the probe is in a flexible region near the surface of the macromolecule and much smaller, less than 0.2, for the rigid sites of the other proteins. For the latter sites the frictional coefficient rises sharply between 40 and 60 degrees C, and its value correlates weakly with molecular parameters such as the depth of burial or the rigidity of a particular site. For RNase T1, coupling to solvent viscosity increases at subzero temperatures, with the coefficient becoming as large as 1 at -20 degrees C. Temperature effects were interpreted by proposing that solvent damping of internal protein motions is particularly effective for low frequency, large amplitude, structural fluctuations yielding highly flexible conformers of the macromolecule.
在探索蛋白质结构的动态特性时，众多研究聚焦于结构波动对溶剂粘度的依赖性，但目前呈现出的情况仍未明确界定。利用色氨酸磷光寿命对其所处环境粘度的敏感性，我们将延迟发射用作蛋白质柔韧性的内在探针，并研究了甘油作为粘性共溶剂的影响。在不同温度下，研究了乙醇脱氢酶、碱性磷酸酶、脱辅基铜蓝蛋白和核糖核酸酶T1的磷光寿命随甘油浓度的变化。关于球状结构中相对刚性位点的柔韧性数据表明，在某些甘油浓度范围内，其对乙醇脱氢酶和核糖核酸酶T1结构波动速率的影响并不遵循关于溶剂粘度的克莱默斯幂律，并强调即使对于大分子的内核，共溶剂诱导的结构变化也可能很重要。当根据克莱默斯模型分析数据时，在0至30摄氏度的温度范围内，对于核糖核酸酶T1，当探针位于大分子表面附近的柔性区域时，得出的摩擦系数相对较大（0.6至0.7），而对于其他蛋白质的刚性位点，摩擦系数则小得多，小于0.2。对于后一种位点，摩擦系数在40至60摄氏度之间急剧上升，其值与诸如埋藏深度或特定位点刚性等分子参数的相关性较弱。对于核糖核酸酶T1，在零下温度下与溶剂粘度的耦合增加，在-20摄氏度时系数高达1。对温度效应的解释是，蛋白质内部运动的溶剂阻尼对于低频、大幅度的结构波动特别有效，从而产生大分子的高度柔性构象。
