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

9314040劳厄以前NSF的支持，这个实验室已经导致了设备的发展，独特的能够直接测量大分子电荷在溶液中，并帮助领导世界上的现代化分析超浓缩。这里概述的工作将联合收割机和扩大这些以前的努力，提供实验手段来检查的作用，静电荷的能量支配rnacromolecular结构和相互作用。测量的电荷和分子结构之间的相关性，产生直接的实验评估：1）的贡献的天然状态的蛋白质的稳定性的电荷，2）的变化，伴随着蛋白质和核酸的结构变化和3）的电荷贡献的能量的大分子协会。为了实现这些目标，需要用于分析超离心和平衡电泳的新仪器和方法。根据这一提议，新的仪器将包括：1）开发用于XLA分析超电泳的荧光检测，2）具有更高精度的平衡电泳装置，其需要更少的时间来达到平衡并且需要更少的样品，3）用于平衡电泳装置的荧光检测能力，以及4）确定和调节导电流体中的小电场的新手段。用于检查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善光谱的去卷积，2）使用生物学产生的光谱增强蛋白质来分析复杂的平衡，3）组合荧光，吸光度和折射率数据，以增加研究的浓度范围，以及4）结合荧光去偏振和沉降速度的结果，以更好地定义分子形状。这些开发的测试系统将由正在进行的合作提供，包括：1）与蛋白质的尿素变性相关的电荷变化，2）电荷在与基因调节相关的蛋白质-蛋白质和蛋白质-核酸相互作用中的作用，3）涉及对血液凝固重要的蛋白质的关联，4）对蛋白聚糖结构和组装重要的大分子相互作用，和5）超螺旋DNA的电荷变化首先松弛，然后线性化。对于第一类，将使用T4溶菌酶和“亮氨酸拉链”肽的电荷突变体。对于这两者，都有良好的天然状态结构，并且变性状态被假定为无规卷曲。在第二类中，我们使用5-OH-葡聚糖光谱增强的cI阻遏物来详细描述蛋白质自缔合和DNA结合之间的联系。 正在启动使用更复杂的CytR-CRP系统的类似研究。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，涉及各种凝血复合物的结构和相互作用的长期项目将继续进行。许多这些蛋白质的特异性荧光标记是可能的，因此荧光光学提供的增加的灵敏度和选择性将允许检查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聚糖组装及其与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、松弛和开放形式时，将测量质粒dsDNA上的电荷，并将其与流体动力学形状和大小相关联。在这些类别中的每一个电荷的知识具有科学的重要性超出仪器的发展。同样，每个测试系统都可以作为一个范例，说明本提案中开发的仪器和方法如何应用于其他类似问题。要求提供资金，以购买设备、用品和技术人员，以便：1）在XLA分析超吸收器上开发荧光检测能力，2）更换/替换圆二色性仪器，3）继续开发和优化平衡电泳设备; 4）继续进行关于平衡电泳的合作实验，并通过沉降和平衡电泳分析异源缔合体系。

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.