Bismuto E, Irace G, D'Auria S, Rossi M, Nucci R
Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Italy.
Eur J Biochem. 1997 Feb 15;244(1):53-8. doi: 10.1111/j.1432-1033.1997.00053.x.
The emission decay of intrinsic fluorescence of the extremely thermophilic beta-glycosidase from Sulfolobus solfataricus has been investigated as functions of temperature and of iodide-quencher concentration by frequency-domain fluorometry. This protein contains 68 tryptophans and provides a matrix for correlation of the average spectroscopic behaviour with solvent exposure and local dynamics. At each temperature, the emission is very heterogeneous and interpretable in terms of quasicontinuous bimodal distribution of fluorescence lifetimes. We associate the component of the bimodal distribution to two distinct classes of tryptophanyl residues that differ in microenvironmental characteristics. Temperature and quenching experiments show that the long-lived component includes tryptophanyl residues located in buried regions with high rigidity; the short distributional component corresponds to tryptophans embedded in more flexible and exposed regions. This proposal has been confirmed by examination of the crystallographic structure. The data suggest that, at least for this protein, there is a good correlation between residue exposure and lifetime distributional components. The conformational dynamics of the two classes of tryptophanyl residues is affected differently by temperature, suggesting that the protein regions in which they are located give different contributions to enzyme properties, such as flexibility, stability and function.
通过频域荧光法研究了嗜热栖热菌中嗜热β-糖苷酶的固有荧光发射衰减与温度和碘化物猝灭剂浓度的关系。这种蛋白质含有68个色氨酸，为将平均光谱行为与溶剂暴露和局部动力学进行关联提供了一个基质。在每个温度下，发射都非常不均匀，并且可以根据荧光寿命的准连续双峰分布来解释。我们将双峰分布的成分与两类微环境特征不同的色氨酸残基联系起来。温度和猝灭实验表明，长寿命成分包括位于刚性较高的埋藏区域的色氨酸残基；短分布成分对应于嵌入更灵活和暴露区域的色氨酸。通过对晶体结构的检查证实了这一推测。数据表明，至少对于这种蛋白质，残基暴露与寿命分布成分之间存在良好的相关性。两类色氨酸残基的构象动力学受温度的影响不同，这表明它们所在的蛋白质区域对酶的性质，如柔韧性、稳定性和功能有不同的贡献。
