Returning to the workforce supplement request for R01 GM095638

返回 R01 GM095638 的劳动力补充请求

• 批准号: 8670457
• 负责人: JENNIFER A MAYNARD
• 金额: $ 7.4万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8670457
• 关键词: Active Sites; Address; Affinity; Alzheimer' s Disease; Antibodies; Aspartate; Binding; Binding Proteins; Biochemical; Bioinformatics; Biological; Biology; Cell surface; Cells; Chemistry; Cocrystallography; Complex; Crystallization; Development; Disease; Distant; Docking; Endoplasmic Reticulum; Engineering; Enzyme-Linked Immunosorbent Assay; Enzymes; Epitopes; Escherichia coli; Gel Chromatography; Health; Hepatitis C; Hepatitis C Therapy; Hepatitis C virus; His-His-His-His-His-His; Homologous Gene; Hydrolysis; Immune response; Immunity; Immunoglobulin Fragments; Knowledge; Location; Major Histocompatibility Complex; Membrane; Membrane Lipids; Membrane Proteins; Methods; Molecular; Molecular Chaperones; Molecular Structure; Muscle Contraction; Mutagenesis; Natural Immunity; Natural Killer Cells; Outcome; Parents; Peptide Hydrolases; Peptide Signal Sequences; Peptides; Phage Display; Process; Proteins; Proteolysis; Randomized; Reagent; Resolution; Role; Route; Signal Transduction; Site-Directed Mutagenesis; Structure; Technology; Therapeutic; Variant; Virus Diseases; Virus Replication; Work; adaptive immunity; directed evolution; experience; immunoregulation; inhibitor/antagonist; interest; new technology; presenilin; protein structure; signal peptide peptidase; virus pathogenesis

Hydrophobic membrane proteins perform a variety of functions in the cell, but their structures are notoriously difficult to solve. Thus, new strategies to obtain crystals of membrane proteins for structure determination are critical. The objectives of this proposal are to develop a toolbox of chaperones and use them to crystallize and solve the de novo, high resolution structure of two signal peptide peptidases (SPPs), which use catalytic aspartates to conduct hydrolysis within the lipid membrane. In contrast to work employing affinity reagents specific to the membrane protein of interest, our potentially transformative approach uses hypercrystallizable single chain antibody fragments (scFvs). Our chaperones are engineered for tight binding to a short epitope that can be inserted into any membrane protein. We expect that our tightly bound scFv chaperone will immobilize an SPP loop and provide a stable crystal lattice, leading to better diffracting crystals. SPPs trim signal peptides (SPs) to liberate them from the endoplasmic reticulum membrane. SPP substrates include SPs remnants derived from new histocompatibility complex 1b (MHC-1b) molecules. As a part of innate immunity, these processed peptides are presented on cell surfaces for recognition by Natural Killer cells to indicate that the cell is healthy. In addition, SPP substrates include SPs from proteins involved in immune response and muscle contraction. SPP is also hijacked by the Hepatitis C virus (HCV) for replication, and is related to presenilin, which uses similar chemistry to generate amyloidogenic peptides in Alzheimer Disease. SPP and presenilin comprise one of just three superfamilies of intramembrane proteases. The details of regulated intramembrane proteolysis, from cell biological signaling to active site chemistry, are of both fundamental biochemical importance and potential therapeutic application. How substrates are presented and hydrolyzed within the confines of the hydrophobic space of the lipid membrane, however, remain largely a mystery. At least 5 SPP variants have been sequenced, located in different regions of ER, and SPPs are conserved throughput biology, but there is no crystal structure yet. We will start by solving the structure an archeal homolog in complex with our chaperones as proof-of principle, and then expand to a eukaryotic SPP, whose biomedical relevant activity is known. To date, we have engineered our first chaperone and isolated an affinity complex with SPP by gel filtration. Independently, we have grown crystals of the chaperone and SPP. However, the crystals of SPP do not diffract well enough for structure determination, and thus the cocrystalllization technology is critical. The expected outcomes are a toolbox of crystallization chaperones as well as the first molecular picture of SPP, including the location of the active site and substrate-docking patches. Taken together, this project will contribute not only to the biology of immunoregulation and intramembrane proteolysis, but also broaden our knowledge of membrane proteins and enable other membrane protein structures to be solved.

疏水膜蛋白在细胞中发挥多种功能，但其结构是出了名的难以解决。因此，获得膜蛋白晶体用于结构测定的新策略是至关重要的。本提案的目标是开发一个伴侣工具箱，并使用它们来结晶和解决两个信号肽肽酶（SPPs）的从头开始，高分辨率结构，它们使用催化天冬氨酸在脂质膜内进行水解。与使用感兴趣的膜蛋白特异性亲和试剂相比，我们潜在的变革方法使用了高结晶单链抗体片段（scFvs）。我们的伴侣蛋白被设计成与短表位紧密结合，可以插入到任何膜蛋白中。我们期望我们紧密结合的scFv伴侣将固定SPP环并提供稳定的晶格，从而产生更好的衍射晶体。