ATOMIC FORCE MICROSCOPY OF BIOPOLYMERS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 BUSM MS Resources进行了原子力显微镜，以补充对单个高分子量生物聚合物的质谱学研究，包括蛋白多糖、胶原蛋白和具有胶原型结构域的分子，以及其他含有高水平糖基化的蛋白质，以及非共价复合体。原子力显微镜正被用来估计这类物种的总重量和模数组成，其基础是在质谱学可获得的范围内建立校准曲线后应能进行的外推。本项目的目的是使用原子力显微镜快速估计大分子和络合物的相对分子质量，作为高分子量范围内的质谱分析的补充工具。这是通过使用AFM测量已知相对分子质量的单分子的分子体积作为标准，使用不同相对分子质量的生物聚合物标准来外推曲线，并确定分子体积与相对分子质量的关系，已经测量了生物聚合物，包括低相对分子质量的蛋白质，如肌红蛋白和白蛋白，高相对分子质量的物种，包括胶原和透明质酸多糖，以及病毒颗粒。体积是使用Berge等人的特别方法从AFM数据估计的。(Berge，T.；Ellis，D.J.；Dryden，D.T.F.；Edwardson，M.J.；Henderson，R.M.生物物理。J.2000、58、1437)。实验结果与Schneider等人的理论计算结果进行了比较。(Schneider，S.W.；Lmer，J.；Henderson，R.M.；Oberleithner，H.；EUR.J.Physiol.1998年435：362)。实验结果表明，尽管在低分子量区，由于针尖尺寸有限而产生的卷积效应，原子力显微镜的测量误差较大，但对于高分子质量标准，测量精度有所提高。虽然需要更多的数据点来获得详细的外推曲线，但这些结果已经表明，AFM是估计高MW范围内尺寸的一种很好的方法。 原子力显微镜也被用来表征淀粉样纤维，目的是将纤维特性与氨基酸序列的变化和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的a-β蛋白，在这一开发项目和几个合作项目(例如，Nugent、Skinner、Spencer、Trinkaus-Randall)中，都在监测不同条件下淀粉样纤维的形成以及蛋白质/GAG相互作用。还记录了从原发性淀粉样病患者的脂肪抽吸物中获得的淀粉样纤维的AFM图像。 仪器的日常操作、维护和监督由洪博士负责，他在波士顿大学系完成了博士学位。细胞生物物理学，重点是粘蛋白的原子力显微镜研究。洪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纤维以及各种大分子的原子力显微镜方面拥有丰富的经验，他帮助规划了这个项目，并继续为BUSM的调查人员提供建议和协助。

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.