Development of Membrane Protein Structure

描述(由申请人提供)：长期的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。这笔资金支持的囊性纤维化跨膜电导调节因子(CFTR)的研究提供了与了解核苷酸结合(NBD)和跨膜(TMD)结构域的结构、导致囊性纤维化(CF)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相关的基本信息。未来的研究将在此基础上详细描述NBD的折叠途径以及CFTR结构域的相互组装和与其他蛋白质结构域在膜上的最终组装。为此，四个特定的目标是：1.表征NBD1的折叠途径，并确定DeltaF508和其他CFs突变的影响。基于cftr-nbd1的高分辨结构，我们将利用生物物理和生化方法来研究折叠途径(S)以及核苷酸突变和cf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。CFTR通道的ABC转运蛋白同源物的核苷酸结合域形成依赖于ATP的二聚体，其中两个核苷酸夹在两个活性ATPase位点上，形成于二聚体界面。我们将检验这样的假设，即cftr nbd1和nbd2形成一个带有一个活跃的ATPase位点和一个不活跃的“调节”位点的ATP夹心二聚体。3.评估NBD和跨膜结构域(TMD)相互作用的能力，并确定导致CF的突变对这些相互作用的影响。F508位置的主链对NBD1折叠至关重要，但侧链对于结构域关联的后续折叠步骤很重要。我们将测试F508位于TMD/NBD1界面的假设。4.评估SLC26A3的STAS结构域与CFTR结合的能力，并确定这对CFTR结构域-结构域结合的影响。为了了解CFTR通道与顶端SLC26a阴离子交换活性的协调调节，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，将结合使用生化、生物物理和细胞生物学方法。这些研究对于详细了解膜蛋白折叠的机制是必要的，也是基本的。

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.