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）：蛋白质构象可能受脂质膜的影响。这一事实似乎是引起HIV辅助蛋白NEF功能的重要因素。 NEF需要与质膜的关联才能下调CD4和MHC受体，从而增强HIV的感染力并促进艾滋病进展。功能齐全的NEF似乎需要从溶液构象过渡到与膜相关的构象。尽管具有明显的疾病重要性，但几乎一无所知。尽管有有关NEF缺失变体的溶液构象的信息，但在功能上重要的环和区域被切除以使该蛋白质与NMR和晶体学兼容。如果可以获得与膜相关和全长NEF的结构细节，则可以更好地理解有关NEF相互作用和随后的生物学作用的基本生物化学。这样的知识基础最终将有助于干扰NEF细胞功能并有可能限制艾滋病的发展和发展的药物的合理设计。研究NEF等蛋白质膜相关构象的替代方法包括氢交换质谱法（HXM）和中子反射仪（NR）。由于HXM和NR可以用少量的稀蛋白和脂质膜进行，因此对于大多数以前的全长NEF的生物物理分析都无法进行条件，我们假设使用这些方法将产生以前无法实现的构象信息。将实现三个具体目标：（1）。了解全长NEF的溶液构象。 HXM将在不同的HIV菌株的NEF上进行。将获得有关全长NEF的缺失构象信息，包括已删除区域的细节以及序列可变性对NEF构象的影响。（2）。确定肉豆蔻酰化的NEF构象在溶液中的不同。将肉豆蔻酰化NEF的HXM与非吡啶并非近去的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HXM将用于探测与膜相关的全长NEF构象，以及与膜锚定靶蛋白（如酪氨酸激酶）结合时。将确定NEF在膜上的总体形状，并将确定结合后的构象变化。综上所述，这些目标有望提供有关以前很难获得的膜相关蛋白质的大量构象信息。由于NEF对于艾滋病毒的感染性至关重要，因此预计这种基本知识将直接适用于未来治疗学的发展。公共卫生相关性：该提案的重点是由称为NEF的人类免疫缺陷病毒（HIV）生产的蛋白质。 NEF对于病毒感染至关重要。这里提出的研究将确定有关该蛋白质形状的信息，尤其是当它与细胞的质膜相互作用时。这种基本知识对于可以干扰NEF功能的治疗剂的开发至关重要，从而防止HIV感染引起艾滋病。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

ETD in a traveling wave ion guide at tuned Z-spray ion source conditions allows for site-specific hydrogen/deuterium exchange measurements.

DOI：
10.1007/s13361-011-0196-7
发表时间：
2011-10
期刊：
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
影响因子：
3.2
作者：
Rand, Kasper D.;Pringle, Steven D.;Morris, Michael;Engen, John R.;Brown, Jeffery M.
通讯作者：
Brown, Jeffery M.

Pepsin immobilized on high-strength hybrid particles for continuous flow online digestion at 10,000 psi.