INVESTIGATING NEUTRALIZING ANTIBODY INDUCED CONFORMATIONAL CHANGES AND MATURATIO

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 对于HIV-1Env蛋白的三聚体形式，几乎没有三维结构信息，现在主要局限于存在一些分歧的低温EM重建。我们已经获得并纯化了多种可溶性、A和B分支HIV-1糖蛋白作为gp140三聚体，可溶性D1D2 CD4和一组中和抗HIV抗体。虽然这些试剂被用于定量结合研究和正在进行的结晶试验，但SAXS可以用于获得补充的结构信息，这些信息可以独立地对我们对抗体中和过程的生物物理基础的研究产生重大影响。在初步收集了SAXS数据后，我们为一个高度敏感的分支B(SF162)gp140三聚体和一个高度抵抗的分支A(Q461)gp140三聚体(加上一系列控制结构)生成了良好的SAXS模型。比较清楚地显示了两者之间的显著构象差异，证明了使用SAXS来检查gp140三聚体的整体结构的可行性。并揭示了三聚体结构可能未被识别的方面。现在，我们提出了一套有凝聚力的实验，使用gp140三聚体的交叉分支小组来绘制gp140亚结构，包括一系列N-连接CHO减少的V-环缺失和形式，一组中和抗体(B12,447-52d，4E10，2F5)和可溶性CD4。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.