Conformational Dynamics of Ribosomal Proteins

[506845] Jameson本研究的目的是阐明和表征核糖体蛋白的构象动力学。待研究的特定系统包括游离的大肠杆菌核糖体蛋白L7/L12和L10，它们被重组为核糖体亚基。通过定点诱变技术，将半胱氨酸残基特别放置在肽链上的适当位置，并用各种巯基特异性荧光探针进行标记。L7/L12和L10都没有固有的色氨酸残基，因此这种固有的荧光团也可以在这些蛋白质的适当位置引入。荧光方法，包括时间分辨和能量转移技术，将被用来阐明L7/L12的不同结构域的灵活性和相对运动，在溶液中，在其五聚体配合物中，L10在50核糖体亚基和完整的70核糖体中。还将研究L7/L12的各种双突变体在缺失推定的铰链区域时的灵活性，以确定这段氨基酸对蛋白质整体构象动力学的贡献。荧光能量转移技术将用于阐明L7/L12上的不同位点之间的距离，包括游离的L7/L12及其与L10的复合物，并重组为核糖体亚基。利用极化和能量转移方法研究L7/L12单体间亚基相互作用的能量学。这些研究将旨在阐明L7/12二聚体/单体平衡的热力学方面，以及研究游离L7/L12溶液及其各种配合物之间的亚基交换。这些生物物理方法将更好地理解L7/L12的亚基相互作用和构象动力学，无论是分离的还是与其他核糖体成分相关的。这些信息将开始提供核糖体的动态描述，这对于详细了解蛋白质生物合成中涉及的协调化学和物理过程最终是必要的。目的是利用荧光技术研究核糖体蛋白的结构和动力学。这些生物物理方法将更好地理解核糖体成分的亚基相互作用和构象动力学。这些信息将开始提供核糖体的动态描述，这对于详细了解蛋白质合成中涉及的协调的化学和物理过程是最终必要的。除了这个研究项目将提供关于一个重要的生物学问题的知识外，它还将通过培养本科生、研究生和博士后，为美国科学教育的基础设施做出贡献。* * * ? ?

9506845 Jameson The goal of this research are to elucidate and characterize the conformational dynamics of ribosomal proteins. The specific systems to be studied include the Escherichia coli ribosomal proteins L7/L12 and L10, free and reconstituted into ribosomal subunits. Cysteine residues, specifically placed at appropriate locations along the peptide chain by site-directed mutagenesis techniques, will be labeled with various sulfhydryl specific fluorescence probes. Neither L7/L12 nor L10 have intrinsic tryptophan residues and hence this intrinsic fluorophore can also be introduced at appropriate locations in these proteins. Fluorescence methods, including time-resolved and energy transfer techniques, will then be utilized to elucidate flexibility and relative motion of different domains of L7/L12, free in solution, in its pentameric complex with L10 in the 50s ribosomal subunit and in the intact 70s ribosome. Flexibility of various double mutants of L7/L12 with deletion of a putative hinge region will also be studied to ascertain the contribution of this stretch of amino acids to the protein's overall conformational dynamics. Fluorescence energy transfer techniques will be utilized to elucidate distances between various sites on L7/L12 both free and in its complexes with L10 and reconstituted into the ribosomal subunits. The energetics of subunit interactions between the L7/L12 monomers will be studied by polarization and energy transfer methods. These studies will be aimed at elucidating the thermodynamic aspects of the dimer/monomer equilibrium of L7/12 as well as studying subunit exchange between L7/L12 free in solution and in its various complexes. These biophysical approaches will afford a better understanding of subunit interactions and conformational dynamics of L7/L12, both isolated and associated with other ribosomal components. This information will begin to provide a dynamic description of the ribosome which is ultimately necessary for detailed understanding of the coord inated chemical and physical processes involved in protein biosynthesis. %%% The goal is to study the structure and dynamics of ribosomal proteins using fluorescence techniques. These biophysical approaches will provide a better understanding of subunit interactions and conformation dynamics of ribosomal components. This information will begin to provide a dynamic description of the ribosome which is ultimately necessary for detailed understanding of the coordinated chemical and physical process involved in protein bi synthesis. In addition to the knowledge this research program will provide on an important biological problem, it will also contribute to the infrastructure of science education in the United States by allowing the training of undergraduate, graduate and postdoctoral students. *** ??