Acquisition of Transient Kinetic Equipment

资金要求购买瞬态动力学设备，用于C）地区州立大学11个不同实验室的研究项目。用户代表四个部门：生物化学和生物物理学，农业化学，药学院和食品科学与技术系。该设备将使用户能够寻求新的方法来解决各自在蛋白质折叠、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-和受体-配体相互作用、酶动力学研究、受体与离子通道的耦合以及线粒体完整性、氧化应激和Ca 2+过载之间的关系等领域的研究问题。 这种设备的可用性应有助于吸引其他研究人员在俄勒冈州州立大学感兴趣的机械研究，因此将作为一个重要的资源教师，博士后，研究生和本科生追求这些研究领域。由于该设备对俄勒冈州来说是新奇的，它也可能吸引来自该州其他机构的用户。该仪器的一个主要特点是数据收集和分析容易。这将有助于用户培训，提高用户在研究中应用动力学和机械方法的积极性。 首席研究员是Michael Schimerlik博士。PI获得了博士学位。和W. W.在威斯康星州的Cleland和瞬态动力学培训，而在加州理工学院的博士后与教授M.A.在那里他进行了配体与烟碱乙酰胆碱受体相互作用的停流研究。自从来到俄勒冈州以来，PI发表了关于蛋白质-蛋白质相互作用和酶机制领域的瞬态动力学研究的手稿，并能够通过将数据拟合到解析导出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（彭纳、货车霍尔德、何、安德森和许博士）也拥有瞬态动力学理论方面的专门知识。 俄勒冈州现有的瞬态动力学设备大约有15年的历史，制造商不再提供支持。没有设施可用于猝灭流实验，以与质谱或NMR研究机构的最新设施结合使用，用于蛋白质折叠研究。没有设施可用于快速采集圆二色谱，以检查发生在蛋白质折叠，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的结构变化。也缺乏获得荧光或吸收光谱作为时间的函数，或测量荧光偏振的时间依赖性变化的能力。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上级目前可用的软件。 总之，拟议的动力学设备将增加重要的新能力，目前在俄勒冈州州立大学不提供瞬态动力学。此外，它将大大增强我们现有的过时设备在所获得的数据类型及其分析方面的能力。这不仅将扩大进行动力学研究的实验室的研究范围，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，使这些人接受瞬态动力学方法的培训。

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.