2D IR OF TRANS-MEMBRANE HELIX STRUCTURES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 二维红外光谱为研究蛋白质的结构和动力学提供了强有力的手段。这种方法可以应用于尚未屈服于传统结构生物学方法的问题。例如，我们最近证明了2D IR可以用来探测膜蛋白中的三级接触。这些实验中研究的蛋白质是血糖素A(GPA)，这是一种在细胞膜上形成结构性同源二聚体的蛋白质。我们现在正在扩展这些方法来研究整合素的跨膜螺旋，整合素是一种参与信号传递和细胞黏附的蛋白质。 跨膜结构域的分子间结合在生理和病理生理过程中都起着重要的作用。越来越多的研究表明，许多跨螺旋的蛋白质的跨膜螺旋所起的作用远远不止是将蛋白质系在膜上。相反，它们经常参与螺旋间的相互作用，这对于指导蛋白质复合体的组装和/或信号转导是必不可少的。近年来，GxxxG基序已成为调节跨膜蛋白相互作用的重要支架。 GxxxG基序(两个Gly残基被任意三个残基分隔)和“GxxxG-like”基序(其中一个或两个Gly残基被Ala或Ser取代的基序)经常被发现对调节TM螺旋之间的相互作用是重要的。在GPA中，GxxxG基序位于L75IxxGVxxGVxxT87二聚体界面的中心。GxxxG基序的凹槽与邻近Val残基的脊相结合，形成一个大的几乎平坦的表面，作为二聚体的中心接触点。围绕这一枢轴点的右手交叉角度似乎受到末端Leu75、Ile76和Thr87残基的限制，这些残基的侧链定位是为了稳定螺旋间几何图形。类似的结构基序经常在其他膜蛋白中观察到。 整合素家族由受体组成，这些受体介导细胞之间以及细胞与细胞外基质之间的双向通讯。整合素是I型整合膜蛋白，在其TM结构域中具有保守的GxxxG样基序，可能参与对信号转导起关键作用的寡聚事件。整合素TM结构域的突变研究表明，GxxxG和GxxxG类似基序主要存在于各种界面的结构中，但尽管在核磁共振和结晶学领域进行了非常广泛的研究，但尚未对该基序进行高分辨率的结构研究。因此，IR将为研究整合素TM螺旋-螺旋相互作用的动力学和结构提供一个重要的工具。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Two-dimensional infrared (2D IR) spectroscopy provides a powerful method to examine protein structure and dynamics. This approach can be applied to problems that have not yet yielded to conventional methods of structural biology. For example, we recently demonstrated that 2D IR can be used to probe for tertiary contacts in a membrane protein. The protein studied in these experiments was glycophorin A (GpA), a protein that forms a constitutive homodimer in membranes. We now are extending these methods to study the transmembrane helices of integrins, proteins that are involved in signaling and cell adhesion. The intermolecular association of transmembrane domains play important roles in physiological as well as pathophysiological processes. Increasingly, it appears that the transmembrane helices of many helix-spanning proteins do far more than tether a protein in a membrane. Instead, they frequently engage in interhelical interactions that are essential for directing the assembly of protein complexes and/or signal transduction. In recent years, the GxxxG motif has emerged as an important scaffold for mediating transmembrane (TM) protein-protein interactions. The GxxxG motif (two Gly residues separated by any three residues), and "GxxxG-like" motifs (motifs in which one or both Gly residues are substituted by Ala or Ser), are often found to be important for mediating interaction of TM helices. In GpA, the GxxxG motif lies within a central in the L75IxxGVxxGVxxT87 dimer interface. The groove of the GxxxG motif, combined with the ridge of the neighboring Val residues, forms a large, almost flat surface, which serves as the central contact point of the dimer. The right-handed crossing angle about this pivot point appears to be constrained by the terminal Leu75, Ile76, and Thr87 residues, whose side chains are positioned so as to stabilize the interhelical geometry. Similar structural motifs are frequently observed in other membrane proteins. The integrin family is composed of receptors that mediate bidirectional communication between cells and between the cell and the extracellular matrix. The integrins are type I integral membrane proteins with a conserved GxxxG-like motif in their TM domains, which are likely to be functionally involved in the oligomerization events that are critical for signaling. Mutagenesis studies of integrin TM domains have shown that GxxxG and GxxxG-like motifs figure largely in the structures of the various interfaces, but there have been no high-resolution structural studies of this motif, despite very extensive efforts in the area of NMR and crystallography. Thus, IR will provide an important tool to examine both the dynamics as well the structures of integrin TM helix-helix interactions.