INVESTIGATING NEUTRALIZING ANTIBODY INDUCED CONFORMATIONAL CHANGES AND MATURATIO

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 对于HIV-1 Env蛋白的三聚体形式，几乎没有三维结构信息，现在主要限于cryoEM重建，其中存在一些分歧。 我们已经鉴定并纯化了多种可溶性的、进化枝A和B HIV-1糖蛋白，如gp 140三聚体、可溶性D1 D2 CD 4和一组中和性抗HIV抗体。虽然这些试剂用于定量结合研究和正在进行的结晶试验，但SAXS可用于获得互补的结构信息，这些信息可独立地对我们研究抗体中和过程的生物物理基础产生重大影响。经过初步的SAXS数据收集，我们产生了良好的SAXS模型的高度敏感的进化枝B（SF 162）gp 140三聚体和高度耐药的进化枝A（Q461）gp 140三聚体（加上一系列的控制结构）。 比较清楚地表明，这两个之间的巨大的构象差异，证明了使用小角X射线衍射检查的gp 140三聚体的整体结构的可行性？并揭示了三聚体结构的潜在未被认识的方面。现在，我们提出了一组连贯的实验，使用gp 140三聚体的交叉进化枝组绘制gp 140亚结构，包括一系列V环缺失和具有还原N-连接CHO的形式，一组中和抗体（b12，447- 52 D，4 E10，2F 5）和可溶性CD 4。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Little three-dimensional structural information is available for trimeric forms of the HIV-1 Env protein, mostly now limited to cryoEM reconstructions where there is some disagreement. We have essed and purified multiple soluble, clade A and B HIV-1 glycoproteins as gp140 trimers, soluble D1D2 CD4 and a panel of neutralizing anti-HIV antibodies. While these reagents are used in quantitative binding studies and ongoing crystallization trials, SAXS can be used to obtain complementary structural information that can independently have significant impact on our studies of the biophysical basis of the process of neutralization by antibodies. After preliminary SAXS data collection, we generated good SAXS models for a highly-sensitive clade B (SF162) gp140 trimer and a highly-resistant clade A (Q461) gp140 trimer (plus a series of control structures). The comparison clearly showed a dramatic conformational difference between these two, demonstrated the viability of using SAXS to examine the overall structure of gp140 trimers ? and revealed potentially unrecognized aspects of trimer structure. Now we propose a cohesive set of experiments to map out gp140 sub-structures using a cross-clade panel of gp140 trimers, including a series of V-loop deletions and forms with reduced N-linked CHO, a set of neutralizing antibodies (b12, 447-52D, 4E10, 2F5) and soluble CD4.