Solid-State NMR of Viral Fusion Peptides and Proteins

描述（由申请人提供）：拟议研究的长期目标是以原子分辨率详细了解病毒融合蛋白诱导膜融合的基础。病毒和靶细胞膜的融合是许多疾病中重要病毒感染的关键步骤，对融合机制的详细了解将有助于开发以融合抑制为作用模式的抗病毒治疗方法。拟建的研究重点分别是人类免疫缺陷病毒（HIV）和流感病毒（IFV）的gp41和血凝素HA2融合蛋白，因为这些病毒引起的疾病具有重要意义，因为这些蛋白可以作为其他I类病毒融合蛋白的原型。特别强调的是融合蛋白的“融合肽”（FP）结构域，它代表了蛋白质n端大约20个残基的极性区域。大量的生物化学和生物物理学研究表明，FP在融合中起着关键作用，具有FP序列的化学合成肽可以作为有用的模型系统来理解病毒/靶细胞融合的某些方面。长期目标将通过四个具体目标来实现。其中两个目标集中在膜中化学交联二聚体和三聚体HIV FPs的固态核磁共振（SSNMR）结构分析和洗涤剂胶束中这些肽的额外液态核磁共振研究。这些c端交联肽的寡聚拓扑结构反映了HIV gp41融合蛋白中FPs的预期拓扑结构。第三个目标侧重于：(a)使用膜在堆叠玻璃板之间取向的样品确定FPs在膜中的插入方向；(b)利用SSNMR测量FP中的13C核与脂质中的31P和19F核之间的距离来探测FP的插入深度。第四个具体目标侧重于包含FPs的HIV和IFV融合蛋白大结构域的SSNMR结构和插入深度测量。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand with atomic-resolution detail the basis for viral fusion protein-induced membrane fusion. Fusion of viral and target cell membranes is a key step in infection for many viruses important in disease and a detailed understanding of fusion mechanisms should aid development of anti-viral therapeutics whose mode of action is fusion inhibition. The proposed research focuses on the gp41 and the hemagglutinin HA2 fusion proteins of the human immunodeficiency virus (HIV) and the influenza virus (IFV), respectively, because of the significance of the diseases caused by these viruses and because these proteins serve as prototypes for other class I viral fusion proteins. There is particular emphasis on the "fusion peptide" (FP) domains of the fusion proteins, which represent approximately 20-residue apolar regions at the N-termini of the proteins. Numerous biochemical and biophysical studies have shown that the FP plays a key role in fusion and that chemically synthesized peptides with the FP sequence can serve as useful model systems to understand some aspects of viral/target cell fusion. The long-term objectives will be achieved with four specific aims. Two of the aims focus on solid-state nuclear magnetic resonance (SSNMR) structural analysis of chemically cross-linked dimeric and trimeric HIV FPs in membranes and additional liquid-state NMR studies of these peptides in detergent micelles. The oligomeric topology of these C-terminal cross-linked peptides reflects the expected topology of FPs in the HIV gp41 fusion protein. The third aim focuses on: (a) determination of the insertion orientation of FPs in membranes using samples in which the membranes are oriented between stacked glass plates; and (b) probing FP insertion depths using SSNMR distance measurements between 13C nuclei in the FP and 31P and 19F nuclei in the lipid. The fourth specific aim focuses on SSNMR structural and insertion depth measurements for large HIV and IFV fusion protein domains which contain FPs.