2D IR OF TRANS-MEMBRANE HELIX STRUCTURES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 二维红外（2D IR）光谱提供了一个强大的方法来检查蛋白质的结构和动力学。 这种方法可以应用于尚未屈服于结构生物学的传统方法的问题。 例如，我们最近证明，二维红外可用于探测膜蛋白中的三级接触。在这些实验中研究的蛋白质是血型糖蛋白A（GpA），一种在膜中形成组成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，整合素是参与信号传导和细胞粘附的蛋白质。 跨膜结构域的分子间缔合在生理和病理生理过程中起着重要作用。 越来越多的研究表明，许多跨螺旋蛋白质的跨膜螺旋不仅仅是将蛋白质拴在膜上。 相反，它们经常参与螺旋间相互作用，这对于指导蛋白质复合物的组装和/或信号转导至关重要。 近年来，GxxxG基序已成为介导跨膜（TM）蛋白质-蛋白质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开）和“GxxxG样”基序（其中一个或两个Gly残基被Ala或Ser取代的基序）通常被发现对于介导TM螺旋的相互作用是重要的。在GpA中，GxxxG基序位于L75 IxxGVxxGVxxT 87二聚体界面的中心。 GxxxG基序的沟与相邻的瓦尔残基的脊结合，形成大的、几乎平坦的表面，其用作二聚体的中心接触点。 关于这个支点的右手交叉角似乎受到限制的终端Leu 75，Ile 76，和Thr 87残基，其侧链的位置，以便稳定的螺旋间的几何形状。 在其他膜蛋白中经常观察到类似的结构基序。 整联蛋白家族由介导细胞之间以及细胞与细胞外基质之间的双向通讯的受体组成。 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，其可能在功能上参与对信号传导至关重要的寡聚化事件。整联蛋白TM结构域的诱变研究表明，GxxxG和GxxxG样图案主要是在各种接口的结构，但一直没有高分辨率的结构研究，尽管在该地区的NMR和晶体学非常广泛的努力，这个主题。 因此，红外光谱将提供一个重要的工具来检查这两个动力学以及整合素TM螺旋-螺旋相互作用的结构。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.