Hansen J E, Steel D G, Gafni A
University of Michigan, Institute of Gerontology, Ann Arbor, 48109-2007 USA.
Biophys J. 1996 Oct;71(4):2138-43. doi: 10.1016/S0006-3495(96)79414-6.
Azurin, a blue copper protein from the bacterial species Pseudomonas aeruginosa, contains a single tryptophan residue. Previous fluorescence measurements indicate that this residue is highly constrained and unusually inaccessible to water. In the apoprotein this residue also possesses a long-lived room-temperature phosphorescence (RTP), the nonexponential decay of which can be resolved into two major components associated with lifetimes of 417 and 592 ms, which likely originate from at least two conformations of the protein. The relative weights of these two decay components change with pH in good correlation with a change in protonation of His-35, which has been studied in Cu(II) azurin. Interestingly, the structural changes characterized in earlier work have little effect on the fluorescence decay and appear to occur away from the tryptophan residue. However, in the present work, the two RTP lifetimes suggest conformations with different structural rigidities in the vicinity of the tryptophan residue. The active conformation that predominates below a pH of 5.6 has the shorter lifetime and is less rigid. Phosphorescence decays of several metal derivatives of azurin were also measured and revealed strong similarities to that of apoazurin, indicating that the structural constraints upon the metal-binding site are imposed predominately by the protein.
天青蛋白是一种来自铜绿假单胞菌的蓝色铜蛋白，含有一个色氨酸残基。先前的荧光测量表明，该残基受到高度限制，且极难与水接触。在脱辅基蛋白中，该残基还具有长寿命的室温磷光（RTP），其非指数衰减可分解为与417和592毫秒寿命相关的两个主要成分，这可能源于该蛋白至少两种构象。这两个衰减成分的相对权重随pH值变化，与已在铜（II）天青蛋白中研究过的His-35质子化变化密切相关。有趣的是，早期工作中表征的结构变化对荧光衰减影响很小，且似乎发生在远离色氨酸残基的地方。然而，在本研究中，两个RTP寿命表明色氨酸残基附近存在具有不同结构刚性的构象。在pH值低于5.6时占主导的活性构象寿命较短且刚性较小。还测量了天青蛋白几种金属衍生物的磷光衰减，结果显示与脱辅基天青蛋白的磷光衰减有很强的相似性，表明对金属结合位点的结构限制主要由蛋白质施加。
