Protein conformational change upon membrane association

膜缔合时蛋白质构象的变化

基本信息

批准号：
8328658
负责人：
JOHN R ENGEN
金额：
$ 38.48万
依托单位：
NORTHEASTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8328658
关键词：
Acquired Immunodeficiency Syndrome Affect Binding Biochemical Biochemistry Biological Biology CD4 Antigens Cell Surface Receptors Cell membrane Cell physiology Cells Country Crystallography Data Development Disease Disease Progression Future Goals HIV HIV-1 Health Hydrogen Indium Knowledge Laboratory Research Length MYO5A gene Mass Spectrum Analysis Measures Membrane Membrane Lipids Membrane Proteins Methods Molecular Conformation Neutrons Outcome Positioning Attribute Property Protein Conformation Protein Tyrosine Kinase Proteins Publishing Research SIV Shapes Signal Pathway Signal Transduction Solutions Structure Structure-Activity Relationship T-Cell Receptor T-Lymphocyte Therapeutic Therapeutic Agents Variant Viral Virus Diseases Work clinically relevant conformational alteration conformational conversion design experience in vivo innovation knowledge base mass spectrometer myristoylation nef Protein prevent receptor therapeutic development therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Protein conformation can be influenced by lipid membranes. This fact seems to be an essential contributor to the function of the HIV accessory protein Nef. Association with the plasma membrane is required for Nef to downregulate CD4 and MHC receptors, thereby enhancing the infectivity of HIV and contributing to AIDS progression. Fully functional Nef appears to require transition from a solution conformation to a membrane-associated conformation. Despite its obvious disease importance, almost nothing is known about the conformation of Nef at the membrane nor about any of Nef's conformational transitions. Although there is information about the solution conformation of Nef deletion variants, functionally important loops and regions were excised to make the protein compatible with NMR and crystallography. If structural details for membrane-associated and full-length Nef could be obtained, the fundamental biochemistry governing Nef interactions and subsequent biological effects would be better understood. Such a knowledge base would ultimately contribute to the rational design of agents that interfere with Nef cellular functions and potentially limit the development of and progression to AIDS. Alternative methods to investigate the membrane-associated conformation of proteins such as Nef include hydrogen exchange mass spectrometry (HXMS) and neutron reflectometry (NR). Because HXMS and NR can be performed with small amounts of dilute protein and in the presence of lipid membranes, conditions not possible with most previous biophysical analyses of full-length Nef, we hypothesize that use of these methods will yield previously unattainable conformational information. Three specific aims will be undertaken: (1). Understand the solution conformation of full-length Nef. HXMS will be performed on Nef from different HIV strains. Missing conformational information about full- length Nef including details of the deleted regions and the effects of sequence variability on Nef conformation will be obtained. (2). Ascertain if and how myristoylated Nef conformation is different in solution. HXMS of myristoylated Nef will be compared to that of non-myristoylated Nef. The regions of conformational alteration upon myristoylation will be determined. (3). Understand the conformation of Nef at the membrane. NR and HXMS will be used to probe the conformation of full-length Nef when associated with membranes and when bound to membrane-anchored target proteins such as tyrosine kinases. The overall shape of Nef at the membrane will be determined and conformational changes upon binding will be ascertained. Taken together, these Aims are expected to provide substantial conformational information about a membrane-associated protein that has been previously very difficult to obtain. As Nef is essential for the infectivity of HIV, this fundamental knowledge is expected to be directly applicable towards the future development of therapeutics. PUBLIC HEALTH RELEVANCE: The focus of this proposal is on a protein made by the human immunodeficiency virus (HIV) called Nef. Nef is essential for viral infectivity. The studies proposed here will determine information about the shape of this protein, especially as it interacts with the plasma membrane of cells. This basic knowledge is essential for the development of therapeutic agents that can interfere with Nef function, thereby preventing HIV infections from causing AIDS.

描述（由申请人提供）：蛋白质构象可能受到脂质膜的影响。这一事实似乎是 HIV 辅助蛋白 Nef 功能的重要贡献者。 Nef 需要与质膜结合才能下调 CD4 和 MHC 受体，从而增强 HIV 的传染性并促进 AIDS 的进展。功能齐全的 Nef 似乎需要从溶液构象转变为膜相关构象。尽管其对疾病的重要性显而易见，但人们对 Nef 在膜上的构象以及 Nef 的任何构象转变几乎一无所知。尽管有有关 Nef 缺失变体的溶液构象的信息，但功能上重要的环和区域被切除，以使蛋白质与 NMR 和晶体学兼容。如果可以获得膜相关和全长 Nef 的结构细节，那么控制 Nef 相互作用和随后的生物效应的基本生物化学将得到更好的理解。这样的知识库最终将有助于合理设计干扰 Nef 细胞功能的药物，并可能限制艾滋病的发展和进展。研究 Nef 等蛋白质膜相关构象的替代方法包括氢交换质谱 (HXMS) 和中子反射测定 (NR)。由于 HXMS 和 NR 可以使用少量稀释蛋白质并在脂膜存在的情况下进行，这是大多数以前的全长 Nef 生物物理分析无法实现的条件，因此我们假设使用这些方法将产生以前无法获得的构象信息。将实现三个具体目标：（1）。了解全长 Nef 的溶液构象。 HXMS 将对来自不同 HIV 毒株的 Nef 进行。将获得有关全长 Nef 的缺失构象信息，包括删除区域的详细信息以及序列变异性对 Nef 构象的影响。（2）。确定肉豆蔻酰化 Nef 构象在溶液中是否不同以及如何不同。将肉豆蔻酰化 Nef 的 HXMS 与非肉豆蔻酰化 Nef 进行比较。将确定肉豆蔻酰化时构象改变的区域。（3）。了解 Nef 在膜上的构象。 NR 和 HXMS 将用于探测全长 Nef 与膜结合以及与膜锚定靶蛋白（如酪氨酸激酶）结合时的构象。 Nef 在膜上的整体形状将被确定，并且结合后的构象变化将被确定。总而言之，这些目标有望提供有关膜相关蛋白的大量构象信息，而这些信息以前很难获得。由于 Nef 对于 HIV 的传染性至关重要，因此这一基础知识预计将直接应用于未来治疗方法的开发。公共健康相关性：该提案的重点是由人类免疫缺陷病毒 (HIV) 产生的一种名为 Nef 的蛋白质。 Nef 对于病毒感染至关重要。这里提出的研究将确定有关这种蛋白质形状的信息，特别是当它与细胞质膜相互作用时。这一基础知识对于开发干扰 Nef 功能的治疗药物至关重要，从而防止 HIV 感染引起艾滋病。