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具有原子分辨率细节的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在疾病中重要的病毒感染的关键步骤，并且对融合机制的详细理解将有助于开发其作用模式为融合抑制的抗病毒治疗剂。融合蛋白也是病毒疫苗开发的目标。拟议的研究重点是人类免疫缺陷病毒和流感病毒各自的gp 41和血凝素HA 2融合蛋白。选择这些蛋白质是因为这些病毒引起主要的人类疾病，并且因为这些蛋白质充当其他I类病毒融合蛋白的原型。特别强调融合蛋白的N-末端“融合肽”（FP）结构域，其代表蛋白质N-末端的~20个残基非极性区域。许多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，化学合成的FP序列肽可以作为有用的模型系统，以了解病毒/靶细胞融合的某些方面。 将通过三个具体目标实现长期目标。目的1集中于生产多mg量的含有FP（包括全长HA 2）的gp 41和HA 2蛋白的大构建体。此外，将制备含有C-末端膜近端外部区（MPER）和病毒跨膜（TM）结构域的gp 41构建体。MPER和TM在融合中是重要的，并且MPER也是HIV疫苗靶点。膜相关构建体的残基特异性构象将通过特定羰基（13 CO）化学位移的固态核磁共振（SSNMR）测量来确定。将特别关注：（1）测试来自短肽研究的FP和MPER的结构模型是否对大构建体中的这些区域有效;（2）将FP和MPER结构与观察到的构建体的融合性相关联;以及（3）检查gp 41 FP是否接近MPER或TM。FP对MPER的阻断可降低MPER作为免疫原的效力。目的2的重点是确定FP，MPER，和TM区域在这些大的gp 41和HA 2结构的膜位置，使用SSNMR测量蛋白质13 CO和脂质或胆固醇31 P，19 F，或2 H之间的距离。这些实验将测试一般结构-功能模型，即深度但非跨膜FP膜插入与更大的膜扰动和更快的融合催化相关。目的3集中于确定B折叠FP在具有非常不同的FP膜位置和融合性的几种gp 41构建体中的反平行登记处的分布。将估计观察到的登记研究的FP膜插入自由能，并将其与膜位置和融合性相关联。总体目标是连接高分辨率FP结构、膜插入深度和融合性。 公共卫生相关性：这项拟议中的研究将提供对人类免疫缺陷病毒和流感病毒进入宿主细胞以及如何阻止这种进入的见解。此外，这项研究还将提供有关如何设计艾滋病毒疫苗的信息。

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.