Development of Membrane Protein Structure

描述（由申请人提供）：长期项目目标是了解折叠信息是如何在膜蛋白序列中编码的，以及细胞识别这一过程失败的方法。该基金支持的囊性纤维化跨膜传导调节因子（CFTR）的研究为理解核苷酸结合（NBD）和跨膜（TMD）结构域的结构、囊性纤维化（CF）引起的折叠突变体的动力学和热力学效应，以及帮助折叠和识别错误折叠CFTR分子的质量控制机制提供了相关的基础信息。未来的研究将在此基础上详细研究NBD的折叠途径，以及CFTR结构域彼此之间以及与膜上其他蛋白质结构域的最终组装。为此目的，有四个具体目标：表征NBD1的折叠途径，并确定deltaF508和其他cfd1突变的影响。基于CFTR-NBD1的高分辨率结构，将采用生物物理和生化方法来表征折叠途径以及核苷酸和cf引起的突变对该途径的影响。2. 评估NBD1和NBD2形成二聚体的能力，并确定cf引起的突变对这些相互作用的影响。ABC转运体同源CFTR通道的核苷酸结合域形成atp依赖二聚体，两个核苷酸夹在二聚体界面形成的两个活性atp酶位点上。我们将检验CFTR NBD1和NBD2形成ATP三明治二聚体的假设，该二聚体具有一个活性ATP酶位点和一个非活性“调节”位点。3. 评估nbd和跨膜结构域（TMDs）相互作用的能力，并确定cf引起的突变对这些相互作用的影响。F508位置的主链对NBD1的折叠至关重要，但侧链对结构域结合的后期折叠步骤也很重要。我们将测试F508位于TMD/NBD1接口的假设。4. 评估SLC26A3的STAS结构域结合CFTR的能力，确定其对CFTR结构域-域关联的影响。为了了解CFTR通道与SLC26A根尖阴离子交换活性的协同调控，我们将阐明SLC26A3的STAS结构域激活CFTR的分子机制。为了实现这四个目标，将采用生物化学、生物物理学和细胞生物学方法的结合。这些研究对于详细了解膜蛋白折叠的机制是必要的和基础的。

DESCRIPTION (provided by applicant): The long-range project objectives are to understand how folding information is encoded in the sequence of membrane proteins and the means by which the cell recognizes failure of this process. Studies of the cystic fibrosis transmembrane conductance regulator (CFTR) supported by this grant have provided fundamental information relevant to understanding the structure of the nucleotide binding (NBD) and transmembrane (TMD) domains, the kinetic and thermodynamic effects of cystic fibrosis (CF)-causing folding mutants, and the quality control mechanisms that assist folding and recognize misfolding CFTR molecules. Future studies build on this foundation by detailing the folding pathway of the NBD and the final assembly of CFTR domains with each other and with domains of other proteins at the membrane. To this end the four specific aims are to: 1. Characterize the folding pathway of NBD1 and determine the effects of the deltaF508 and other CFmutations. Building on the high resolution structure of CFTR-NBD1, biophysical and biochemical methods will be employed to characterize the pathway(s) of folding and the effects of nucleotide and CF-causing mutations on the pathway. 2. Assess the ability of NBD1 and NBD2 to form dimers and determine the effect of CF-causing mutations on these interactions. Nucleotide binding domains of ABC transporter homologues of the CFTR channel form ATP-dependent dimers, with two nucleotides sandwiched at two active ATPase sites formed at the dimer interface. We will test the hypothesis that CFTR NBD1 and NBD2 form an ATP sandwich dimer with a single active ATPase site and an inactive "regulatory" site. 3. Assess the ability of the NBDs and the transmembrane domains (TMDs) to interact and determine the effect of CF-causing mutations on these interactions. The backbone at the F508 position is critical for NBD1 folding, but the side chain is important for the later folding steps of domain association. We will test the hypothesis that F508 lies at the TMD/NBD1 interface. 4. Assess the ability of the STAS domain of SLC26A3 to bind CFTR and determine the effect of this on CFTR domain-domain association. To understand the coordinated regulation of CFTR channel and apical SLC26A anion exchange activity, we will elucidate the molecular mechanism by which the STAS domain of SLC26A3 activates CFTR. To accomplish these four goals, a combination of biochemical, biophysical, and cell biological approaches will be employed. These studies are necessary for and fundamental to a detailed understanding of the mechanisms by which membrane proteins fold.