DEVELOPMENT OF HIGH-THROUGHPUT PULSE DIPOLAR ESR SPECTROSCOPY (HT-PDS)

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 随着PDS在生物医学研究中应用的迅速扩大，该领域遇到了有限的吞吐量的障碍。为了获得所需的结构和功能机制的细节，通常必须在广泛的实验条件下筛选和研究许多（大约一百个）样品。我们在ACERT开发了一种非常灵敏和可靠的Ku波段脉冲光谱仪，十年来可以说是PDS最灵敏的光谱仪。它还具有执行两个主要PDS脉冲序列的独特能力：DEER（双电子-电子共振）和DQC。 在ACERT，我们一直在解决如何在P41 ACERT核心项目I“脉冲ESR蛋白质结构测定”下显著提高PDS通量的问题。 NIH的补充资金将使我们能够在未来两年内实现这一重要目标。我们的计划，这是在我们最初的建议中概述，是增加吞吐量的一个因素，我们目前在ACERT实现我们的高灵敏度Ku波段脉冲ESR光谱仪，使足够的样品（高达约100）可以在一天的数据采集研究，这通常是足够完成一个特定的结构/功能研究。因此，例如，灵敏度增加一个数量级将使数据收集时间减少100倍，即建立完整的三角测量网络仅需一天时间，并在此基础上确定蛋白质结构。这通常足以解析二级结构的元素，以确定三级结构，和/或将蛋白质对接到蛋白质复合物中。这将满足用于解决结构的高通量（HT）技术的标准，具有每年解决数十个（或可能数百个）蛋白质的能力，仅受限于产生大量突变体的能力。我们计划开发的基本方法是以高灵敏度并行处理大量样品。开发HT PDS的计划包括几个具体目标，这些目标在随后的七个按时间顺序排列（但必然相互交织）的单独子项目（0276-0282）中详细说明。这些目标是：（1）开发一种系统，用于在非常低的温度下使用批处理自动采集连续样品的数据;（2）通过提高工作频率和优化探头设计来提高脉冲光谱仪的灵敏度;（3）实现低温接收器，其在低温下操作以提供非常低的噪声温度，从而进一步提高灵敏度，从而缩短数据采集时间;（4）构建一个在10-70 K的温度范围内连续运行的无冷冻剂系统;（5）扩展自动数据采集，以同时处理多个样品，从而显著提高产量;（6）提高样品处理和加工的效率;（7）提高PDS数据处理效率。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. With the rapidly expanding growth of applications of PDS in biomedical study, the field is encountering the roadblock of limited throughput. Often many (of the order of a hundred) samples have to be screened and studied under a wide range of experimental conditions in order to obtain the desired structure and details of the functional mechanisms. We have developed at ACERT a very sensitive and reliable pulse spectrometer operating at Ku band, which for a decade was arguably the most sensitive spectrometer anywhere for PDS. Also it has the unique capability of performing both of the main PDS pulse sequences: DEER (double electron-electron resonance) and DQC. At ACERT we have been addressing the issue of how to increase the throughput for PDS significantly under P41 ACERT Core Project I: "Protein Structure Determination by Pulse ESR". NIH supplementary funding is to enable us to achieve this important goal within the next two years. Our plans, which were outlined in our original proposal, are to increase throughput by a factor of 25 to 100 over what we currently achieve at ACERT on our high sensitivity Ku-band pulse ESR spectrometer, so that sufficient samples (up to about 100) could be studied in a single day of data acquisition, and this would typically be sufficient to complete a particular structure/function study. Thus, for example, an increase in sensitivity by one order of magnitude would reduce the data collection time by a factor of 100, i.e. it would be just a matter of a day to build a full triangulation network, upon which to determine the protein structure. This often suffices to resolve elements of secondary structure, to determine tertiary structure, and/or dock proteins into protein complexes. This would meet the criterion of high throughput (HT) technology for solving structures, with the capability of solving tens (or maybe hundreds) of proteins per year, limited only by the ability to produce large number of mutants. The basic approach that we plan to develop is to have parallel processing of a large number of samples with high sensitivity. The plan to develop HT PDS encompasses several specific aims, which are detailed in the subsequent seven time-ordered (but necessarily intertwined) individual subprojects (0276-0282). These aims are: (1) develop a system for automated data acquisition of consecutive samples at very low temperatures using batch processing; (2) increase the sensitivity of the pulse spectrometer by increasing the working frequency and optimizing the probe-head design; (3) implement a cryogenic receiver operating at a low temperatures to provide a very low noise temperature to further increase the sensitivity, thereby shortening data acquisition time; (4) construct a cryogen-free system that will operate continuously at temperatures in the range of 10-70 K; (5) extend the automated data acquisition to handle several samples simultaneously, thereby significantly increasing throughput; (6) improve the efficiency of sample handling and processing; (7)improve PDS data processing efficiency.