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了解病毒融合蛋白诱导膜融合的原子分辨率细节的基础。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。融合蛋白也是病毒疫苗开发的目标。本研究拟重点研究人类免疫缺陷病毒和流感病毒的gp41和血凝素HA2融合蛋白。之所以选择这些蛋白质，是因为这些病毒会导致主要的人类疾病，而且这些蛋白质可以作为其他I类病毒融合蛋白的原型。特别强调融合蛋白的n端“融合肽”（FP）结构域，它代表了蛋白质n端约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。长期目标将通过三个具体目标来实现。目的1主要研究含有FPs的gp41和HA2蛋白大构建体（包括全长HA2）的多mg量的产生。此外，gp41构建物将包含c端膜近端外区（MPER）和病毒跨膜（TM）结构域。MPER和TM在融合中起重要作用，MPER也是HIV疫苗的靶点。膜相关结构的残基特异性构象将通过固态核磁共振（SSNMR）测量特定羰基（13CO）化学位移来确定。将特别关注：(1)测试来自短肽研究的FP和MPER结构模型是否在大型结构中对这些区域有效；(2)将FP和MPER结构与观察到的构建体的融合性联系起来；(3)检测gp41 FP是否接近MPER或TM。FP阻断MPER可降低MPER作为免疫原的作用。目的2侧重于利用sssnmr测量蛋白13CO与脂质或胆固醇31P、19F或2H之间的距离，确定这些大型gp41和HA2构建体中FP、MPER和TM区域的膜位置。这些实验将测试深层而非跨膜FP膜插入与更大的膜扰动和更快的融合催化相关的一般结构-功能模型。目的3的重点是确定几个具有不同FP膜位置和融合性的gp41构建体中b片FP的反平行注册表的分布。FP膜插入的自由能将被估计为观察到的注册，并与膜的位置和融合性相关。总体目标是连接高分辨率FP结构，膜插入深度和融合性。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand the basis for viral fusion protein-induced membrane fusion with atomic-resolution detail. 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 will aid development of anti-viral therapeutics whose mode of action is fusion inhibition. Fusion proteins are also a target of viral vaccine development. The proposed research focuses on the gp41 and the hemagglutinin HA2 fusion proteins of the respective human immunodeficiency virus and influenza virus. These proteins are chosen because the viruses cause major human diseases and because the proteins serve as prototypes for other class I viral fusion proteins. There is particular emphasis on the N-terminal "fusion peptide" (FP) domains of the fusion proteins, which represent ~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 three specific aims. Aim 1 focuses on production of multi-mg quantities of large constructs of gp41 and HA2 proteins that contain FPs, including full-length HA2. In addition, gp41 constructs will be made which contain the C-terminal membrane-proximal external region (MPER) and viral transmembrane (TM) domains. MPER and TM are important in fusion and MPER is also an HIV vaccine target. Residue-specific conformation of the membrane-associated constructs will be determined with solid- state nuclear magnetic resonance (SSNMR) measurements of specific carbonyl (13CO) chemical shifts. There will be particular focus on: (1) testing whether structural models of FP and MPER from short peptide studies are valid for these regions in the large constructs; (2) correlating FP and MPER structure to the observed fusogenicity of the construct; and (3) examining whether the gp41 FP is close to either the MPER or TM. Occlusion of MPER by FP could reduce MPER efficacy as an immunogen. Aim 2 focuses on determining the membrane locations of FP, MPER, and TM regions in these large gp41 and HA2 constructs using SSNMR measurements of distances between protein 13CO and lipid or cholesterol 31P, 19F, or 2H. These experiments will test the general structure-function model that deep but not transmembrane FP membrane insertion correlates with greater membrane perturbation and more rapid fusion catalysis. Aim 3 focuses on determining the distribution of antiparallel registries of b sheet FP in several gp41 constructs with very different FP membrane locations and fusogenicities. The free energies of FP membrane insertion will be estimated for the observed registries and correlated with membrane locations and fusogenicities. The overall goal is to connect high-resolution FP structure, membrane insertion depth, and fusogenicity. PUBLIC HEALTH RELEVANCE: The proposed research will provide insight into entry of human immunodeficiency virus and influenza virus into host cells and how this entry can be stopped. In addition, the research will provide information about how a HIV vaccine should be designed.