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

9314040 Laue先前NSF对该实验室的支持导致了能够直接测量溶液中大分子电荷的独特设备的开发，并帮助引领了世界分析超离心机的现代化。这里概述的工作将结合和扩展这些先前的努力，提供实验手段来检查静电电荷在决定分子结构和相互作用的能量学中的作用。将在测量的电荷和分子结构之间建立相关性，从而产生以下方面的直接实验评估：1)电荷对蛋白质天然状态稳定性的贡献；2)伴随着蛋白质和核酸结构变化的电荷变化；3)电荷对核糖分子结合的能量学的贡献。为了实现这些目标，需要新的仪器和方法来分析超离心和平衡电泳。该提案将产生的新仪器包括：1)开发用于XLA分析超离心机的荧光检测；2)具有更高精度的平衡电泳装置，达到平衡所需的时间更短，需要的样品更少；3)平衡电泳装置的荧光检测能力；4)确定和调节导电流体中的小电场的新方法。改进的检测分子关联的方法将包括：1)在线测定消光系数和荧光量子产率以改善光谱的反褶积；2)利用生物产生的光谱增强蛋白来分析复杂的平衡态；3)结合荧光、吸光度和折射数据来增加所研究的浓度范围；4)结合荧光去极化和沉降速度的结果来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，涉及：1)与尿素变性相关的蛋白质电荷变化，2)与基因调控相关的蛋白质-蛋白质和蛋白质-核酸相互作用中的电荷作用，3)涉及血液凝固重要蛋白质的关联，4)对蛋白聚糖结构和组装重要的大分子相互作用，以及5)超卷曲DNA首先放松，然后线性化时的电荷变化。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。对于两者，良好的自然状态结构是可用的，变性状态被假定为一个随机线圈。在第二类中，我们使用5-OH-色氨酸光谱增强的cI抑制因子来详细描述蛋白质自结合与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，荧光光学提供的灵敏度和选择性增加，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，工作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，当分子处于超卷曲、松弛和开放状态时，将测量平面dsDNA上的电荷，并与流体动力学形状和大小相关。这些类别中的电荷的知识具有超出仪器开发的科学重要性。同样，每个测试系统都可以作为范例，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。为以下方面所需的设备、用品和技术人员寻求资金：1)开发XLA分析超离心机的荧光检测能力；2)翻新/更换圆二色仪；3)继续开发和优化平衡电泳设备；4)继续进行正在进行的平衡电泳和通过沉淀和平衡电泳分析异质缔合体系的合作实验。

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.