Acquisition of Transient Kinetic Equipment

需要资金从C)地区州立大学的11个不同实验室购买用于研究项目的瞬变动力学设备。用户代表四个系：生物化学和生物物理系、农业化学系、药学院和食品科学与技术系。该设备将允许用户在蛋白质折叠、蛋白质-蛋白质和蛋白质-DNA相互作用、DNA和蛋白质结构、蛋白质与受体配体相互作用、酶的动力学研究、受体与离子通道的偶联以及线粒体完整性、氧化应激和钙超载之间的关系等领域寻求新的方法来解决各自的研究问题。这种设备的提供应该有助于吸引俄勒冈州立大学对机械研究感兴趣的其他研究人员，因此将成为从事这些研究领域的教师、博士后、研究生和本科生的重要资源。由于这种设备对俄勒冈州来说是新的，它也可能吸引该州其他机构的用户。该工具的一个主要特点是数据收集和分析的简便性。这应有助于用户培训，并提高用户在其研究中采用动态、机械性方法的积极性。首席调查员是迈克尔·希默里克博士。PI在威斯康星州获得了W.W.Cleland教授的酶动力学博士学位，并接受了瞬时动力学方面的培训，同时在加州理工学院与M.A.Raftery教授一起做博士后研究，在那里他对配体与尼古丁乙酰胆碱受体的相互作用进行了停流研究。自从来到俄勒冈州立大学以来，PI已经发表了关于蛋白质-蛋白质相互作用和酶机制领域的瞬时动力学研究的手稿，并能够通过将数据拟合到解析推导的速率方程来分析动力学机制，或者在更复杂的情况下，使用计算机模拟来检查机制模型。其他几位研究人员(Penner、van Holde、Ho、Anderson和Hsu博士)也拥有瞬变动力学理论方面的专业知识。俄勒冈州立大学现有的瞬变动力设备大约有15年的历史，不再受到制造商的支持。没有设备可用于急流实验，以便与研究所最先进的质谱学或核磁共振设备结合使用，以研究蛋白质折叠。没有设备可以快速获取圆二色谱来检查蛋白质折叠、配体结合、DNA和蛋白质结构转变或大分子组装或解离反应中发生的结构变化。还缺乏获取作为时间函数的荧光或吸收光谱的能力，或测量荧光偏振随时间变化的能力。这些技术提供了额外的灵敏度，并增强了对复杂机制的分析。最重要的是，数据采集和分析软件远远优于目前可用的软件。总之，拟议的动力学设备将增加俄勒冈州立大学目前没有的瞬变动力学方面的重要新能力。此外，它还将在获取的数据类型及其分析方面大大增强我们目前可用的过时设备的能力。这不仅将扩大进行动力学研究的实验室的研究视野，而且由于其“用户友好性”，有助于吸引博士后、研究生和本科生，使这些人能够接受瞬时动力学方法的培训。

Funds are requested to purchase transient kinetic equipment to be employed in research projects from eleven different laboratories at C)region State University. The users represent four departments: Biochemistry and Biophysics, Agricultural Chemistry, the College of Pharmacy and the Department of Food Science and Technology. The equipment will allow the users to pursue new approaches to their respective research problems in the areas of protein folding, protein-protein and protein-DNA interactions, DNA and protein structure, protein- and receptorligand interactions, kinetic studies of enzymes, the coupling of receptors to ion channels and the relationship between mitochondrial integrity, oxidative stress and Ca2+ overload. The availability of this equipment should serve to attract other researchers at Oregon State University interested in mechanistic studies and will therefore serve as an important resource for faculty, postdoctorals, graduate students and undergraduates pursuing these research areas. Since the equipment is novel to Oregon, it may also attract users from other institutions within the state. One major feature of the instrumentation is the ease of data collection and analysis. This should facilitate user training and increase the enthusiasm of users in applying kinetic, mechanistic approaches in their research. The principal investigator is Dr. Michael Schimerlik. The PI received his Ph.D. in enzyme kinetics with Professor W.W. Cleland at Wisconsin and training in transient kinetics while a postdoctoral at The California Institute of Technology with Professor M.A. Raftery where he conducted stopped-flow studies of ligand interactions with the nicotinic acetylcholine receptor. Since coming to Oregon State, the PI has published manuscripts concerning transient kinetic studies in the areas of protein-protein interactions and enzyme mechanisms and is capable of analysis of kinetic mechanisms by fitting data to analytically derived rate equations or, i n more complicated instances, using computer simulations to examine mechanistic models. Several other investigators (Drs. Penner, van Holde, Ho, Anderson and Hsu) also possess expertise in transient kinetic theory. Transient kinetic equipment existing at Oregon State is about fifteen years old, and no longer supported by the manufacturer. No facilities are available for quench flow experiments for use in combination with state of the art facilities at the institution in mass spectrometry or NMR for studies of protein folding. No facilities are available for rapid acquisition of circular dichroism spectra to examine structural changes that occur in protein folding, ligand binding, DNA and protein structural transitions, or macromolecular assembly or dissociation reactions. The abilities to acquire fluorescence or absorbance spectra as a function of time, or to measure time-dependent changes in fluorescence polarization are also lacking. These techniques provide additional sensitivity and enhance the analysis of complicated mechanisms. Most importantly, the software for data acquisition and analysis is far superior to that currently available. In summary, the proposed kinetic equipment will add important new capabilities in transient kinetics not currently available at Oregon State University. In addition it will provide greatly enhanced capabilities over that of our currently available, outdated equipment both in the type of data acquired and in its analysis. This will not only expand the research horizons of laboratories doing kinetic studies but, also because of its "user-friendliness", serve to attract postdoctorals, graduate students and undergraduates so that these individuals will receive training in transient kinetic methods.