Pittman I, Tager H S
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637, USA.
Biochemistry. 1995 Aug 22;34(33):10578-90. doi: 10.1021/bi00033a033.
A conformational change, termed the T --> R transition, which can be detected by visible, circular dichoric, and fluorescence spectroscopy, occurs in native insulin and tryptophan substituted insulin analogs ([TrpB25]-, [TrpB26]-, [GlyB24,TrpB25]-, and [GlyB24,TrpB26]insulin) upon binding specific alcohol ligands, including phenol and cyclohexanol. In these studies we have demonstrated that changes in the visible absorbance spectrum of an insulin6(Co2+)2 solution are not a definitive means of determining the occurrence of T --> R transitions in the presence of alcohol ligands. We also have presented evidence that fast protein liquid chromatography (FPLC) can be used to determine the aggregation state of insulin and that des-octapeptide(B23-30)insulin (DOI) forms Zn(2+)-coordinated hexamers that appear to be stabilized by the T --> R transformation. Using fluorescence spectroscopy, we have shown that in the presence of specific alcohol ligands the B-chain COOH-terminal residues, particularly position B25, of hexameric, as well as monomeric insulin undergo a conformational change which appears to be related to the T --> R transformation. Circular dichroic studies indicate that a conformation similar to the R-state of metal-coordinated hexameric insulin can be induced by binding cyclohexanol; however, this new conformational state (RI-state) exists independent of divalent metal ion coordination, and therefore of hexamer formation. We further show that monomeric insulin can be induced to assume the RI-state upon alcohol binding, therefore illustrating the first defined conformational change described for monomeric insulin. We suggest that this new conformation may be an intermediate state in the T --> R transformation in metal-coordinated hexameric insulin, such that T --> RI --> R. The model presented here of the structural adjustments undergone by insulin upon binding cyclohexanol provides further insight into the conformational flexibility of insulin in solution.
一种被称为T→R转变的构象变化，可通过可见光谱、圆二色光谱和荧光光谱检测到，在天然胰岛素和色氨酸取代的胰岛素类似物（[TrpB25]-、[TrpB26]-、[GlyB24,TrpB25]-和[GlyB24,TrpB26]胰岛素）与特定醇类配体（包括苯酚和环己醇）结合时发生。在这些研究中，我们已经证明，在存在醇类配体的情况下，胰岛素6(Co2+)2溶液可见吸收光谱的变化并不是确定T→R转变发生的决定性方法。我们还提供了证据表明，快速蛋白质液相色谱（FPLC）可用于确定胰岛素的聚集状态，并且去八肽（B23 - 30）胰岛素（DOI）形成Zn(2+)配位的六聚体，这些六聚体似乎通过T→R转变而稳定。使用荧光光谱法，我们已经表明，在存在特定醇类配体的情况下，六聚体以及单体胰岛素的B链COOH末端残基，特别是B25位，会发生构象变化，这似乎与T→R转变有关。圆二色性研究表明，通过结合环己醇可以诱导出与金属配位的六聚体胰岛素的R态相似的构象；然而，这种新的构象状态（RI态）独立于二价金属离子配位而存在，因此也独立于六聚体形成。我们进一步表明，单体胰岛素在结合醇类后可被诱导呈现RI态，因此说明了单体胰岛素所描述的第一个明确的构象变化。我们认为这种新构象可能是金属配位的六聚体胰岛素中T→R转变的中间状态，即T→RI→R。这里提出的胰岛素结合环己醇时所经历的结构调整模型，进一步深入了解了胰岛素在溶液中的构象灵活性。
