Molecular Structure Determination by Mass Spectrometry and Computational Modeling

通过质谱和计算模型测定分子结构

基本信息

批准号：
10735319
负责人：
Joshua S Sharp
金额：
$ 48.6万
依托单位：
UNIVERSITY OF MISSISSIPPI
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-06-01 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10735319
关键词：
Address Amino Acids Antibody Binding Sites Antithrombin III Area Binding Biological Sciences Blood Blood Proteins Buffers Carbohydrate Chemistry Carbohydrates Cell model Cells Chromatography Complex Complex Mixtures Computer Analysis Computer Models Computer Simulation Coupled Data Development Dose Emerging Technologies Engineering Epitopes Family Freezing Generations Health Heparin High Pressure Liquid Chromatography Histones Human Hydrogen Peroxide Hydrophobic Interactions Hydroxyl Radical Immune response Immunoglobulin G In Situ In Vitro Individual Ion Exchange Ions Label Life Liquid Chromatography Mass Spectrum Analysis Measurement Measures Mediating Membrane Proteins Methods Modernization Modification Molecular Molecular Conformation Molecular Structure Monoclonal Antibodies Mus Organelles Ovalbumin Patients Pharmaceutical Preparations Pharmacology Plasma Play Polysaccharides Post-Translational Protein Processing Protein Analysis Protein Conformation Protein Dephosphorylation Protein Footprinting Protein Isoforms Protein Structure Databases Proteins Proteome Reagent Reporting Reproducibility Research Personnel Resolution Role Side Site Solvents Structure Surface System TNF gene Techniques Technology Testing Time Tissues Whole Blood Work adalimumab biophysical properties carbohydrate structure cell injury chemical stability conformer dosimetry dynamic system experience experimental study flexibility high standard improved in vivo innovation mechanical force molecular mechanics molecular modeling mouse model protein aggregation protein complex protein structure reaction rate structural biology therapeutic protein three dimensional structure tool tool development

项目摘要

PROJECT SUMMARY/ABSTRACT Structural biology plays a central role in modern molecular bioscience, enabling both a greater understanding and new mechanisms of manipulation of biomolecular action. However, despite tremendous development in tools for the generation of high resolution molecular models, large families of biomolecules and biomolecular complexes are still poorly represented in databases of protein structure due to limitations of current technology, and methods for probing protein structure within mammalian tissue are few. One method that has been used successfully to qualitatively study the structure of several of these families is hydroxyl radical protein footprinting (HRPF), an emerging technology that has been used to study changes in protein topography by measuring changes in the apparent rate of reaction between hydroxyl radicals generated in situ and amino acid side chains on the protein surface. Our initial work has developed HRPF into a quantitative measurement of protein topography at the individual amino acid level, accurately measuring the average solvent accessible surface areas (<SASA>) of many individual amino acids in a single experiment. In this renewal, we will expand our technology into structural systems that change dynamically with time, including protein posttranslational modification systems, large heteromeric protein complexes, and protein:carbohydrate complexes. The core technology we will develop to enable these studies is high performance liquid chromatography coupled inline with amino acid resolution HRPF (LC-HR-HRPF). Inline liquid chromatography allows the separation of protein conformers and immediate quantitative measurement of the purified conformers’ topographies by HR-HRPF before the dynamic system has a chance to re-equilibrate, freezing the structural information in the stable chemical footprint. We will also develop technology for analysis of protein structure within mammalian whole blood, enabling the study of protein structure and interactions within highly complex native systems. We will develop flow systems to precisely and carefully deliver hydrogen peroxide to blood for protein labeling without damaging cells, and will demonstrate the technology with the structural analysis of monoclonal antibodies dosed into a mouse model. Finally, we will develop technologies to probe the topography of complex carbohydrates, enabling us to measure which parts of carbohydrates mediate interactions with proteins, even in complex mixtures of glycans. We will develop both reducing-end specific and non-specific labeling strategies for probing carbohydrate topography. Together, these advances represent potential transforming technologies for the structural analysis of biomedically important and highly challenging systems.

项目概要/摘要结构生物学在现代分子生物科学中发挥着核心作用，使然而，尽管有巨大的进展，但人们对生物分子作用的理解和操纵的新机制。开发用于生成高分辨率分子模型、生物分子大家族和由于生物分子复合物的局限性，生物分子复合物在蛋白质结构数据库中的代表性仍然很差。目前探测哺乳动物组织内蛋白质结构的技术和方法很少。已成功用于定性研究其中几个家族的结构的是羟基自由基蛋白质足迹（HRPF），一种用于研究蛋白质变化的新兴技术通过测量原位产生的羟基自由基之间的表观反应速率的变化来测量形貌我们的初步工作已将 HRPF 发展为定量方法。测量单个氨基酸水平的蛋白质形貌，准确测量平均值在此实验中，许多单独的氨基酸的溶剂可及表面积（<SASA>）。更新，我们将把我们的技术扩展到随时间动态变化的结构系统中，包括蛋白质翻译后修饰系统、大型异聚蛋白质复合物和蛋白质：碳水化合物我们为实现这些研究而开发的核心技术是高性能液体。与氨基酸分辨率 HRPF 联用的色谱法 (LC-HR-HRPF)。允许分离蛋白质构象异构体并立即定量测量纯化结果在动态系统有机会重新平衡之前，通过 HR-HRPF 调整构象，冻结我们还将开发蛋白质分析技术。哺乳动物全血中的结构，使得能够研究高度内的蛋白质结构和相互作用我们将开发流动系统，以精确、小心地将过氧化氢输送至复杂的原生系统。血液进行蛋白质标记而不损伤细胞，并将展示该技术的结构最后，我们将开发探测技术。复杂碳水化合物的拓扑结构，使我们能够测量碳水化合物的哪些部分介导与蛋白质的相互作用，甚至在复杂的聚糖混合物中，我们将开发还原端特异性和特异性。这些进步代表了探测碳水化合物拓扑的非特异性标记策略。用于生物医学重要且极具挑战性的结构分析的潜在转化技术系统。

项目成果

期刊论文数量（19）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The structure of a virus-encoded nucleosome.

DOI：
10.1038/s41594-021-00585-7
发表时间：
2021-05
期刊：
Nature structural & molecular biology
影响因子：
16.8
作者：
Valencia-Sánchez MI;Abini-Agbomson S;Wang M;Lee R;Vasilyev N;Zhang J;De Ioannes P;La Scola B;Talbert P;Henikoff S;Nudler E;Erives A;Armache KJ
通讯作者：
Armache KJ

Intrinsic Buffer Hydroxyl Radical Dosimetry Using Tris(hydroxymethyl)aminomethane.

DOI：
10.1021/jasms.9b00088
发表时间：
2020-02-05
期刊：
Journal of the American Society for Mass Spectrometry
影响因子：
3.2
作者：
Roush AE;Riaz M;Misra SK;Weinberger SR;Sharp JS
通讯作者：
Sharp JS

Peracylation Coupled with Tandem Mass Spectrometry for Structural Sequencing of Sulfated Glycosaminoglycan Mixtures without Depolymerization.

DOI：
10.1021/jasms.0c00178
发表时间：
2020-10-07
期刊：
Journal of the American Society for Mass Spectrometry
影响因子：
3.2
作者：
Liu H;Liang Q;Sharp JS
通讯作者：
Sharp JS

Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes.

DOI：
10.3791/61580
发表时间：
2020-09-01
期刊：
Journal of visualized experiments : JoVE
影响因子：
0
作者：
Misra SK;Sharp JS
通讯作者：
Sharp JS

Cryo-EM reveals the architecture of placental malaria VAR2CSA and provides molecular insight into chondroitin sulfate binding.

DOI：
10.1038/s41467-021-23254-1
发表时间：
2021-05-19
期刊：
Nature communications
影响因子：
16.6
作者：
Wang K;Dagil R;Lavstsen T;Misra SK;Spliid CB;Wang Y;Gustavsson T;Sandoval DR;Vidal-Calvo EE;Choudhary S;Agerbaek MØ;Lindorff-Larsen K;Nielsen MA;Theander TG;Sharp JS;Clausen TM;Gourdon P;Salanti A
通讯作者：
Salanti A