ATOMIC FORCE MICROSCOPY OF BIOPOLYMERS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 BUSM MS资源已经进行了原子力显微镜，以补充高分子量的单个生物聚合物的质谱研究，包括，例如，蛋白聚糖、胶原和具有胶原型结构域的分子，以及含有高水平糖基化的其它蛋白质，以及非共价复合物。原子力显微镜正被用来估计这些物种的总重量和模块组成，外推的基础上，应该成为可能后，建设校准曲线的范围内可通过质谱。本项目的目的是利用原子力显微镜快速估计大分子和复合物的分子量，作为质谱在高分子量范围内的补充工具。这是通过使用AFM来测量已知MW的单分子的分子体积作为标准，使用不同MW的生物聚合物标准外推曲线，并确定分子体积和MW之间的关系来实现的。已经测量了生物聚合物，包括低分子量蛋白质如肌红蛋白和白蛋白，包括胶原蛋白和多糖透明质酸的较高MW物质，以及病毒颗粒。 使用Berge等人的特别方法（Berge，T.; Ellis，D. J.道：德莱登，D. T. F.地; Edwardson，M. J.道：亨德森河M. Biophys. J. 2000，58，1437）。实验结果与Schneider等人的理论计算进行了比较（Schneider，S. W的; Lomer，J.;亨德森河M.; Oberleithner，H.; EUR. 1998 435：362）。实验结果表明，虽然在低分子量范围内，AFM测量给出了一个相对较大的误差，由于卷积效应所造成的有限的针尖尺寸，精度增加高分子量标准。 虽然需要更多的数据点来获得详细的exprapolation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。 原子力显微镜也被用来表征淀粉样蛋白原纤维，与原纤维的性质有关的氨基酸序列的变化和翻译后修饰的组成蛋白质的目标。 通过使用纯化的患者样品以及重组α-β蛋白，在本开发项目和几个合作项目（例如，Nugent，Skinner，Spencer，Trinkaus-Randall）.从原发性淀粉样疾病患者的脂肪抽吸物中获得的淀粉样纤维也记录了AFM图像。 仪器的日常操作、维护和监督由洪博士负责，他在波士顿大学医学系完成了博士学位。细胞生物物理学的重点是粘蛋白的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蛋白纤维和各种类型的大分子的AFM方面拥有丰富的经验，他帮助规划了这个项目，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.