ATOMIC FORCE MICROSCOPY OF BIOPOLYMERS

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 BUSM MS Resource 采用原子力显微镜来补充单个高分子量生物聚合物的质谱研究，包括蛋白聚糖、胶原蛋白和具有胶原蛋白型结构域的分子，以及含有高水平糖基化的其他蛋白质以及非共价复合物。 AFM 正用于估计此类物种的总重量和模块组成，其基础是在质谱法可达到的范围内构建校准曲线后应该成为可能的外推法。该项目的目的是使用 AFM 快速估算大分子和复合物的分子量，作为高分子量范围内质谱分析的补充工具。这是通过使用 AFM 测量已知 MW 的单分子的分子体积作为标准，使用不同 MW 下的生物聚合物标准品推断曲线，并确定分子体积与 MW 之间的关系来实现的。生物聚合物，包括低分子量蛋白质，如肌红蛋白和白蛋白，较高分子量的物种，包括胶原蛋白和多糖透明质酸，以及病毒颗粒，都已被测量。 使用 Berge 等人的特别方法根据 AFM 数据估计体积。 (Berge, T.；Ellis, D. J.；Dryden, D. T. F.；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。 (Schneider, S.W.；Lomer, J.；Henderson, R.M.；Oberleithner, H.；Eur. J. Physiol. 1998 435:362)。实验结果表明，尽管在低分子量范围内，由于有限尖端尺寸引起的卷积效应，AFM测量给出了相对较大的误差，但对于高分子量标准，精度有所提高。 尽管需要更多数据点才能获得详细的推算曲线，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 还用于表征淀粉样原纤维，目的是将原纤维特性与氨基酸序列的变化以及组成蛋白的翻译后修饰联系起来。 通过使用纯化的患者样本以及重组 α-β 蛋白，在该开发项目和几个合作项目（例如 Nugent、Skinner、Spencer、Trinkaus-Randall）中，正在监测不同条件下淀粉样原纤维的形成以及蛋白质/GAG 相互作用。还记录了从患有原发性淀粉样蛋白疾病的患者的脂肪抽吸物中获得的淀粉样蛋白原纤维的 AFM 图像。 仪器的日常操作、维护和监督由 Hong 博士负责，他在波士顿大学细胞生物物理学系获得了博士学位，重点是粘蛋白的 AFM 研究。 Hong 博士对仪器的整体性能进行了评估，并获得了几类生物聚合物的图像，在空气和水/缓冲液中进行了实验。 丁博士在β-淀粉样蛋白和突触核蛋白淀粉样原纤维以及各种类型大分子的AFM方面拥有丰富的经验，他帮助规划了该项目，并继续为BUSM研究人员提供建议和帮助。

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.