ATOMIC FORCE MICROSCOPY OF BIOPOLYMERS

生物聚合物的原子力显微镜

• 批准号: 7722947
• 负责人: Catherine E. Costello
• 金额: $ 1.81万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722947
• 关键词: Air; Albumins; Amino Acid Sequence; Amyloid Fibrils; Amyloid beta-Protein; Amyloidosis; Aspirate substance; Atomic Force Microscopy; Biophysics; Biopolymers; Boston; Buffers; Calibration; Class; Collagen; Complement; Complex; Computer Retrieval of Information on Scientific Projects Database; Condition; Data; Development; Doctor of Philosophy; Evaluation; Facility Construction Funding Category; Fatty acid glycerol esters; Funding; Goals; Grant; Hyaluronan; Image; Individual; Institution; Maintenance; Mass Spectrum Analysis; Measurement; Measures; Molecular; Molecular Weight; Monitor; Mucins; Myoglobin; Operative Surgical Procedures; Patients; Performance; Polysaccharides; Post-Translational Protein Processing; Property; Proteins; Proteoglycan; Range; Recombinants; Research; Research Personnel; Resources; Sampling; Source; Standards of Weights and Measures; Supervision; United States National Institutes of Health; Universities; Variant; Virion; Water; Weight; amyloid fibril formation; base; day; experience; glycosylation; instrument; macromolecule; research study; single molecule; size; synuclein; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The BUSM MS Resource has undertaken atomic force microscopy to complement mass spectrometry studies of individual biopolymers of high molecular weight, including, e.g., proteoglycans, collagens and molecules having collagen-type domains, and other proteins that contain high levels of glycosylation, as well as non-covalent complexes. AFM is being used to estimate the overall weight and modular composition of such species, on the basis of extrapolations that should become possible after construction of calibration curves over the range accessible by mass spectrometry. The aim of this project is to use AFM to quickly estimate the MWs of large molecules and complexes as a supplemental tool for mass spectrometry in the high molecular weight range. This is achieved by using AFM to measure the molecular volume of single molecules of known MW as standards, extrapolating a curve using biopolymer standards at different MW, and determining the relation between molecular volume and MW, Biopolymers, including low molecular weight proteins such as myoglobin and albumin, higher MW species including collagen and the polysaccharide hyaluronan, and virus particles, have been measured. The volume is estimated from the AFM data using the ad hoc approach of Berge et al. (Berge, T.; Ellis, D. J.; Dryden, D. T. F.; Edwardson, M. J.; Henderson, R. M. Biophys. J. 2000, 58, 1437). The experimental results were compared with theoretical calculation by Schneider et al. (Schneider, S. W.; Lomer, J.; Henderson, R. M.; Oberleithner, H.; Eur. J. Physiol. 1998 435:362). The experimental results show that although, in the low MW range, the AFM measurements give a relatively large error due to the convolution effect caused by the finite tip size, the accuracy increases for high molecualr weight standards. Although more data points are required to achieve a detailed exprapolation curve, these results have already shown that AFM is a good way to estimate size in the high MW range. AFM is also being used to characterize amyloid fibrils, with the goal of relating fibril properties to variations in the amino acid sequence and to posttranslational modifications of the constituent proteins. By using purified patient samples as well as recombinant a-beta protein, the formation of amyloid fibrils under different conditions and protein/GAG interactions is being monitored in both this development project and in several collaborative projects (e.g., Nugent, Skinner, Spencer, Trinkaus-Randall). AFM images have also been recorded for amyloid fibrils obtained from fat aspirates of patients with primary amyloid disease. Day-to-day operation, maintenance and supervision of the instrument are the responsibility of Dr. Hong, who completed his PhD in the Boston University Dept. of Cellular Biophysics with a focus on AFM studies of mucins. Dr. Hong has carried out evaluations of overall instrument performance and has obtained images for several classes of biopolymers, performing experiments in both air and water/buffer. Dr. Ding, who has extensive experience in AFM of beta-amyloid and synuclein amyloid fibrils and various types of macromolecules, helped in the planning for this project and continues to provide advice and assistance to the BUSM investigators.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 BUSM MS资源已经进行了原子力显微镜，以补充高分子重量的个别生物聚合物的质谱研究，包括，例如，蛋白聚糖，胶原蛋白和具有胶原蛋白型结构域的蛋白聚糖，胶原蛋白和分子，以及其他含有高水平的糖基化蛋白质的蛋白质，以及非甘油基化的高水平。 AFM被用来估计此类物种的总体重量和模块化组成，基于推断，在构建校准曲线在质谱范围内构建校准曲线后应成为可能。该项目的目的是使用AFM快速估计大分子和复合物的MW作为高分子重量范围内质谱法的补充工具。 This is achieved by using AFM to measure the molecular volume of single molecules of known MW as standards, extrapolating a curve using biopolymer standards at different MW, and determining the relation between molecular volume and MW, Biopolymers, including low molecular weight proteins such as myoglobin and albumin, higher MW species including collagen and the polysaccharide hyaluronan, and virus particles, have been测量。 使用Berge等人的临时方法从AFM数据中估算该体积。 （Berge，T。; Ellis，D。J。; Dryden，D。T.; Edwardson，M.J。; Henderson，R。M.Biophys。J.2000，58，1437）。 Schneider等人将实验结果与理论计算进行了比较。 （施耐德（S.实验结果表明，尽管在低MW范围内，AFM测量结果由于有限的尖端尺寸引起的卷积效应而产生的误差相对较大，但高分子重量标准的准确性会提高。 尽管需要更多的数据点才能实现详细的挖掘曲线，但这些结果已经表明，AFM是估计高MW范围内尺寸的好方法。 AFM还用于表征淀粉样蛋白原纤维，其目的是将原纤维性质与氨基酸序列的变化和成分蛋白的翻译后修饰有关。 通过使用纯化的患者样品以及重组A-BetA蛋白，在此开发项目和几个协作项目中都在监测不同条件下的淀粉样蛋白原纤维和蛋白质/GAG相互作用（例如Nugent，Skinner，Spinner，Spencer，Spencer，Trinkaus-Randall）。还记录了从原发性淀粉样蛋白疾病患者的脂肪抽吸物获得的淀粉样蛋白原纤维的记录。 该工具的日常运作，维护和监督是Hong博士的责任，他在波士顿大学的蜂窝生物物理学部完成了博士学位，重点是对粘蛋白的AFM研究。 Hong博士对整体仪器性能进行了评估，并获得了几类生物聚合物的图像，并在空气和水/缓冲液中进行了实验。 Ding博士在AFM拥有β-淀粉样蛋白和突触核蛋白淀粉样蛋白原纤维以及各种类型的大分子的AFM经验，他为该项目的计划提供了帮助，并继续为Busm的调查人员提供建议和帮助。