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）：艾滋病毒疫苗发育的最流行和有希望的靶标之一是源自GP120的免疫原子，GP120是一种包膜糖蛋白，这是病毒进入CD4细胞所必需的。最近，我们的合作者分离了多种HIV对GP120的抗体广泛中和抗体，并表明它们需要某些N连接的聚糖进行活动。但是，其中一些抗体没有结合自由的N连接甘氨酸，这表明其中至少有些也可能与GP120的蛋白质部分相互作用。抗体。GP120复合物太大而无法通过NMR光谱产生高分辨率结构，并且由于聚糖似乎在抗体表位中起关键作用，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，但是纯计算方法可以生成可疑准确性的模型，并且需要经验约束或测试以生成可靠的模型。我们建议使用GP120-抗体复合物的羟基自由基蛋白足迹来表征各种广泛中和抗体的表位，该技术基于其溶剂的可及性，该技术标记了各种氨基酸侧链。为了提高足迹数据对准确，高分辨率模型构建的实用性，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准确的定量，以改善结构分辨率和归一化协议，以产生足迹数据中的绝对溶剂溶剂可访问的表面积。我们还建议开发适当的评分函数，以利用溶剂可访问的表面区域作为分子动力学模拟的约束，类似于使用距离约束。从这些改进及其应用到GP120抗体复合物的表征，我们预计会产生准确的，实验约束的模型，这些模型正确地识别了每种抗体的表位。这些模型对于免疫原子的合理设计将非常重要，以通过免疫来提高宿主中相应的广泛中和抗体。 公共卫生相关性：艾滋病毒仍然是全世界，尤其是发展中国家的毁灭性疾病。尽管需要迫切需要，但向艾滋病毒的疫苗开发仍然难以捉摸。最近，通过与包膜糖蛋白GP120相互作用来中和各种HIV血清型的一系列抗体已从HIV阳性患者中分离出来。这些抗体要求聚糖结合GP120，但至少其中一些不仅与聚糖结合。但是，抗体识别的表位尚未被定义，因此不可能通过疫苗开发免疫原以提高宿主中的抗体。我们建议利用羟基自由基足迹，加上计算建模来表征和建模GP120与每种广泛中和抗体之间的复合物。这些模型将有助于免疫原子的合理发展，以制定有效的抗HIV疫苗。