Improved Hydroxyl Radical Footprinting for Modeling Protein Structure

改进的羟基自由基足迹用于蛋白质结构建模

• 批准号: 8236656
• 负责人: Joshua S Sharp
• 金额: $ 26.37万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8236656
• 关键词: Affinity; Algorithms; American; Amino Acid Sequence; Amino Acids; Antibodies; Antigens; Area; Binding; CD4 Positive T Lymphocytes; Cells; Comparative Study; Complex; Computer Simulation; Coupled; Crystallization; Data; Developing Countries; Development; Diffuse; Disease; Epitopes; Europe; Funding; Generations; Glycoproteins; HIV; HIV Envelope Protein gp120; HIV Seropositivity; HIV vaccine; HIV-1; Hydroxyl Radical; Immunization; Infection; Isomerism; Label; Laboratories; Ligand Binding; Ligands; Link; Measurement; Measures; Methods; Modeling; Monitor; Monoclonal Antibodies; NMR Spectroscopy; Patients; Peptide Sequence Determination; Peptides; Play; Polysaccharides; Process; Protein Footprinting; Proteins; Protocols documentation; Publishing; Research; Research Personnel; Resolution; Series; Serotyping; Serum; Side; Site; Solvents; Structural Models; Structure; Surface; Techniques; Technology; Testing; Time; Vaccines; Viral; Virus; Work; base; design; improved; interest; member; molecular dynamics; neutralizing antibody; neutralizing monoclonal antibodies; oxidation; prevent; protein structure; protein structure prediction; research study; restraint; tandem mass spectrometry; vaccine development

DESCRIPTION (provided by applicant): One of the most popular and promising targets for HIV vaccine development are immunogens derived from gp120, an envelope glycoprotein essential for viral entry into CD4 cells. Recently, our collaborators have isolated multiple HIV broadly neutralizing antibodies to gp120 and shown that they require certain N-linked glycans for activity. However, some of these antibodies do not bind the free N-linked glycans, suggesting that at least some of them may also interact with the protein portion of gp120. The antibody.gp120 complexes too large for generation of high-resolution structures by NMR spectroscopy, and as the glycan appear to play key roles in the antibody epitopes, crystallization of the complexes is a daunting task. Computational modeling is an attractive approach for this problem, but purely computational approaches can generate models of questionable accuracy, and require empirical constraints or testing in order to generate a reliable model. We propose to characterize the various broadly neutralizing antibody epitopes using hydroxyl radical protein footprinting of the gp120-antibody complexes, a technique that labels a broad variety of amino acid side chains based on their accessibility to solvent. In order to improve the usefulness of the footprinting data for accurate, high-resolution model building, we propose to develop a number of improved footprinting methods, including accurate quantitation at the amino acid level to improve structural resolution and normalization protocols to generate absolute solvent accessible surface area values from footprinting data. We also propose to develop an appropriate scoring function to utilize solvent accessible surface areas as a constraint in molecular dynamics simulations, analogous to the use of distance constraints. From these improvements and their application to the characterization of gp120-antibody complexes, we anticipate the generation of accurate, experimentally-constrained models that correctly identify the epitope for each antibody. These models will be very important for the rational design of immunogens to raise the corresponding broadly neutralizing antibodies in a host through immunization. PUBLIC HEALTH RELEVANCE: HIV remains a devastating disease throughout the world, especially in developing countries. Despite the urgent need, development of a vaccine to HIV remains elusive. Recently, a series of antibodies that neutralize a broad variety of HIV serotypes through interaction with the envelope glycoprotein gp120 have been isolated from HIV-positive patients. These antibodies require the glycan to bind gp120, but at least some of them do not bind solely to the glycan. However, the epitope that the antibody recognizes has not been defined, so it is not possible to develop an immunogen to raise the antibody in a host through a vaccine. We propose to utilize hydroxyl radical footprinting coupled with computational modeling to characterize and model the complex between gp120 and each of the broadly neutralizing antibodies. These models will aid in the rational development of immunogens to formulate effective anti-HIV vaccines.

描述（由申请人提供）：HIV 疫苗开发中最受欢迎和最有前途的目标之一是源自 gp120 的免疫原，gp120 是病毒进入 CD4 细胞所必需的包膜糖蛋白。最近，我们的合作者分离出了多种针对 gp120 的 HIV 广泛中和抗体，并表明它们需要某些 N 连接聚糖才能发挥活性。然而，其中一些抗体不结合游离的 N 连接聚糖，这表明它们中至少一些也可能与 gp120 的蛋白质部分相互作用。抗体.gp120 复合物太大，无法通过 NMR 光谱生成高分辨率结构，并且由于聚糖似乎在抗体表位中发挥关键作用，因此复合物的结晶是一项艰巨的任务。计算建模是解决此问题的一种有吸引力的方法，但纯粹的计算方法可能会生成准确性有问题的模型，并且需要经验约束或测试才能生成可靠的模型。我们建议使用 gp120-抗体复合物的羟自由基蛋白足迹来表征各种广泛中和的抗体表位，该技术根据氨基酸侧链对溶剂的可及性来标记多种氨基酸侧链。为了提高足迹数据在准确、高分辨率模型构建中的有用性，我们建议开发许多改进的足迹方法，包括在氨基酸水平上进行精确定量以提高结构分辨率和标准化协议以生成绝对溶剂可访问的来自足迹数据的表面积值。我们还建议开发适当的评分函数，以利用溶剂可及的表面积作为分子动力学模拟中的约束，类似于距离约束的使用。从这些改进及其在 gp120-抗体复合物表征中的应用，我们预计会生成准确的、实验约束的模型，以正确识别每种抗体的表位。这些模型对于合理设计免疫原以通过免疫在宿主体内产生相应的广泛中和抗体非常重要。 公共卫生相关性：艾滋病毒仍然是全世界的一种毁灭性疾病，特别是在发展中国家。尽管迫切需要，但艾滋病毒疫苗的开发仍然难以实现。最近，从 HIV 阳性患者中分离出一系列通过与包膜糖蛋白 gp120 相互作用来中和多种 HIV 血清型的抗体。这些抗体需要聚糖来结合 gp120，但至少其中一些抗体不仅仅与聚糖结合。然而，抗体识别的表位尚未确定，因此不可能开发出免疫原通过疫苗在宿主体内产生抗体。我们建议利用羟基自由基足迹与计算模型相结合来表征和模拟 gp120 与每种广泛中和抗体之间的复合物。这些模型将有助于合理开发免疫原，以配制有效的抗艾滋病毒疫苗。