Protein conformational change upon membrane association

膜缔合时蛋白质构象的变化

• 批准号: 7935460
• 负责人: JOHN R ENGEN
• 金额: $ 39.34万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7935460
• 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; 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; public health relevance; receptor; therapeutic development; therapeutic target

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.

性状（由申请方提供）：蛋白质构象可受脂膜影响。这一事实似乎是HIV辅助蛋白Nef功能的重要贡献者。Nef需要与质膜结合以下调CD4和MHC受体，从而增强HIV的感染性并促进AIDS进展。全功能Nef似乎需要从溶液构象转变为膜相关构象。尽管其明显的疾病的重要性，几乎没有什么是已知的构象的Nef在膜上也没有任何的Nef的构象转换。虽然有关于Nef缺失变体的溶液构象的信息，但切除功能上重要的环和区域以使蛋白质与NMR和晶体学相容。如果能获得膜相关和全长Nef的结构细节，将更好地理解Nef相互作用和随后的生物效应的基本生物化学。这样的知识基础最终将有助于合理设计干扰Nef细胞功能并可能限制艾滋病发展和进展的药物。研究蛋白质（如Nef）的膜相关构象的替代方法包括氢交换质谱法（HXMS）和中子反射法（NR）。由于HXMS和NR可以进行少量的稀释蛋白质和脂质膜的存在下，不可能与大多数以前的生物物理分析全长Nef的条件下，我们假设，使用这些方法将产生以前无法获得的构象信息。具体目标有三：（1）。理解全长Nef的溶液构象。将对不同HIV毒株的Nef进行HXMS。将获得关于全长Nef的缺失构象信息，包括缺失区域的细节和序列变异性对Nef构象的影响。（二）、确定肉豆蔻酰化Nef构象在溶液中是否不同以及如何不同。将豆蔻酰化Nef的HXMS与非豆蔻酰化Nef的HXMS进行比较。豆蔻酰化后构象改变的区域将被确定。（三）、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。将确定Nef在膜上的整体形状，并确定结合后的构象变化。两者合计，这些目标预计将提供大量的构象信息的膜相关蛋白质，以前很难获得。由于Nef对HIV的感染性至关重要，因此预计这一基础知识将直接适用于未来的治疗发展。公共卫生相关性：该提案的重点是人类免疫缺陷病毒（HIV）产生的一种称为Nef的蛋白质。Nef对病毒的传染性至关重要。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。这些基本知识对于开发能够干扰Nef功能的治疗剂，从而预防HIV感染引起艾滋病至关重要。