Instruments and Methods for Determining the Role of Charge in Macromolecular Structure and Assembly

9314040劳厄之前美国国家科学基金会对该实验室的支持导致了独特的能够直接测量溶液中大分子电荷的设备的开发，并帮助引领了分析超速离心机的现代化。这里概述的工作将结合和扩展这些先前的工作，以提供实验手段来检查静电电荷在决定大分子结构和相互作用的能量学中的作用。测量的电荷和分子结构之间的关联将产生直接的实验评估：1)电荷对蛋白质天然状态稳定性的贡献，2)伴随蛋白质和核酸结构变化的电荷变化，以及3)电荷对大分子缔合能量学的贡献。为了实现这些目标，需要用于分析超速离心法和平衡电泳法的新的仪器和方法。这一建议带来的新仪器将包括：1)开发XLA分析超速离心机的荧光检测；2)更高精度的平衡电泳设备，达到平衡所需的时间和样品更少；3)平衡电泳设备的荧光检测能力；以及4)确定和调节导电流体中小电场的新方法。研究大分子缔合的改进方法将包括：1)在线测定消光系数和荧光量子产率，以改进光谱的去卷积；2)使用生物产生的光谱增强蛋白质来分析复杂的平衡；3)结合荧光、吸光度和折射数据以扩大研究的浓度范围；4)结合荧光去极化和沉积速度的结果以更好地确定分子形状。这些发展的测试系统将由正在进行的合作提供，涉及：1)与蛋白质尿素变性有关的电荷变化，2)与基因调控有关的蛋白质-蛋白质和蛋白质-核酸相互作用中电荷的作用，3)涉及对凝血重要的蛋白质的关联，4)对蛋白多糖结构和组装重要的大分子相互作用，以及5)随着超螺旋DNA首先松弛，然后线性化的电荷变化。对于第一类，将使用T4溶菌酶和亮氨酸拉链多肽的电荷突变体。对于两者，良好的自然状态结构都是可用的，并且假设变性状态是随机卷曲。在第二类中，我们使用了5-OH-色氨酸光谱增强的Ci抑制物来详细说明蛋白质自结合和DNA结合之间的联系。对更复杂的CytR-CRP系统的类似研究正在启动。需要荧光检测来表征这些系统中的紧密结合事件。在第三类中，涉及各种凝血复合体的结构和相互作用的长期项目将继续进行。对这些蛋白质中的许多进行特定的荧光标记是可能的，因此荧光光学提供的更高的灵敏度和选择性，将允许检测更高的亲和力和更复杂的相互作用。在第四类中，将继续收集有关蛋白多糖组装及其与多肽激素相互作用的基本信息。在最后一类中，当分子处于超螺旋、松弛和开放状态时，将测量分子dsDNA上的电荷，并与流体动力学形状和大小相关联。这些类别中的每一种电荷的知识具有超越仪器开发的科学重要性。同样，每个测试系统都是如何将本提案中开发的工具和方法应用于其他类似问题的范例。为以下目的寻求资金：1)开发XLA分析超速离心机的荧光检测能力，2)翻新/更换圆二色谱仪器，3)继续开发和优化平衡电泳仪，4)继续进行关于平衡电泳法和通过沉淀和平衡电泳法分析异质缔合体系的持续合作实验。

9314040 Laue Previous NSF support of this lab has resulted in the development of devices uniquely capable of measuring macromolecular charge directly in solution and has helped lead the world in the modernization of the analytical ultracentrifuge. The work outlined here will combine and expand these previous efforts to provide the experimental means to examine the role of electrostatic charge in the energetics dictating rnacromolecular structures and interactions. Correlations will be made between measured charge and molecular structures, yielding direct experimental assessments of: 1) the contribution of charge to the stability of the native state of proteins, 2) the change in charge that accompanies structural changes in proteins and nucleic acids and 3) the charge contribution to the energetics of rnacromolecular associations. To accomplish these goals, new instrumentation and methodologies for analytical ultracentrifugation and equilibrium electrophoresis are needed. New instrumentation resulting from this proposal will include: 1) the development of fluorescence detection for the XLA analytical ultracentrifuge, 2) equilibrium electrophoresis devices with greater accuracy, that require less the time to reach equilibrium and need less sample, 3) fluorescence detection capabilities for equilibrium electrophoresis devices, and 4) a new means of determining and regulating small electric fields in a conducting fluid. Improved rnethods for examining rnacromolecular associations will include: 1) on- line determination of extinction coefficients and fluorescence quantum yields to improve the deconvolution of spectra, 2) the use of biologically-produced spectrally- enhanced protein for the analysis of complicated equilibria, 3) combining fluorescence, absorbance and refractive data to increase the concentration range studied and 4) combining results from fluorescence depolarization and sedimentation velocity to better define molecular shapes. Test systems for thes e developrnents will be provided by on-going collaborations involving: 1) the change in charge associated with urea denaturation of proteins, 2) the role of charge in protein-protein and protein-nucleic acid interactions relating to gene regulation, 3) associations involving proteins important to blood coagulation, 4)macromolecular interactions important to proteoglycan structure and assembly, and 5) the change in charge as supercoiled DNA is first relaxed, then linearized. For the first category, charge mutants of T4 lysozyme and "leucine zipper" peptides will be used. For both, good native-state structures are available, and the denatured state is presumed to be a random coil. In the second category, we have used 5-OH- tryptophan-spectrally-enhanced cI repressor to detail the linkage between protein self association and DNA binding. Similar studies with the more complicated CytR-CRP system are being initiated. Fluorescence detection is needed to characterize the tight binding events in these systems. In the third category, long-standing projects involving the structures and interactions of various blood coagulation complexes will continue. Specific fluorescent labeling of many of these proteins is possible, so that the increased sensitivity and selectivity afforded by fluorescence optics, will allow the examination of both higher affinity and more complicated interactions. In the fourth category, work will continue on gathering basic information concerning proteoglycan assembly and their interactions with peptide hormones. In the last category, the charge on plamid dsDNA will be measured when the molecule is in supercoiled, relaxed and open forms, and correlated with hydrodynamic shape and size. Knowledge of the charge in each of these categories has scientific importance beyond the instrumentation development. Likewise, each test system serves as a paradigm for how the instruments and methods developed in this proposal can be applied to other, similar prob lems. Funds are sought for the equipment, supplies and technical personnel needed to: 1) develop fluorescence detection capabilities on the XLA analytical ultracentrifuge, 2) refurbish/replace a circular dichroism instrument, 3) continue the development and optimization of equilibrium electrophoresis equipment and 4) continue ongoing collaborative experiments on equilibrium electrophoresis and the analysis of heteroassociating systems by sedimentation and equilibrium electrophoresis.