Folding of a Multidomain Ribosomal Protein

阐明氨基酸序列如何决定结构，即蛋白质折叠问题，是现代结构生物学的核心问题。这一建议描述了一种综合的方法来研究蛋白质L9的折叠，一个多结构域核糖体蛋白。该蛋白形成了一个有趣的双结构，紧凑的n端结构域由一个长溶剂暴露的a-螺旋连接到紧凑的c -末端结构域。n端结构域是一个稳定的折叠单元，代表了一类重要的层状片状螺旋结构的简单例子之一。这种结构存在于其他核糖体蛋白和许多小RNA结合域中。相对而言，人们对稳定这种重要结构的相互作用以及它如何折叠知之甚少。L9蛋白也为研究多结构域蛋白的折叠提供了一个很好的模型系统。近年来，人们对单域球形蛋白的折叠途径进行了大量的研究，但对多域球形蛋白的折叠途径却知之甚少。该蛋白的折叠途径将通过动力学测量来探索。分离结构域和完整L9蛋白的平衡和动力学折叠跃迁将被研究。这些实验将表明结构域之间的相互作用是否会影响蛋白质的折叠或稳定性。展开态将用核磁共振和圆二色性来表征。对肽片段的研究将用于解决局部相互作用在稳定n端结构域中的作用。这些研究还将确定任何潜在的折叠起始位点。片段互补实验将用于探索三级相互作用在稳定新生二级结构中的作用。综上所述，这些实验将提供折叠途径的详细描述以及稳定这种有趣蛋白质的相互作用。在一所大型大学教学的主要挑战之一是平衡本科教育与研究。许多学生觉得他们“在混乱中迷失了方向”，或者认为本科教育远远落后于研究和研究生教育。另一方面，现有的研究基础设施有可能为本科生提供独特的机会。该职业奖的教育方面包括一些旨在将本科生融入该系研究生活的举措。正在开设一门新课程“生物化学的最新趋势”。本课程旨在向本科生介绍现代生物化学的研究领域，并围绕系内生物化学系列研讨会展开。一个非正式的系列研讨会，旨在向学生介绍许多可供化学家和生物化学家选择的职业。作为本科核心课程之一的物理化学实验课程正在进行广泛的修订和重新定义。引入了新的、更相关的实验，并采用了更多的互动教学方法。在研究生阶段，正在设计一个新的生物化学课程。这包括一个新的系列研讨会，这也是“生物化学的当前趋势”课程的基础；在化学系设立一个新的学位选择，设计一个关于蛋白质结构的新课程，并编写一个跨部门培训补助金。阐明氨基酸序列如何决定结构，即蛋白质折叠问题，是现代结构生物学的核心问题。本文介绍了一种研究多结构域核糖体蛋白L9折叠的综合方法。对于单域球形蛋白的折叠途径的研究已经取得了相当大的进展，但对于多域球形蛋白的折叠途径却知之甚少。这个分子的孤立结构域也很有趣。n端结构域是一个稳定的折叠单元，是核糖体蛋白和小RNA结合结构域中发现的一种重要结构的简单例子之一。在一所大型大学教学的主要挑战之一是平衡本科教育与研究。许多学生觉得他们“在混乱中迷失了方向”，本科教育远远落后于研究和研究生教育。该职业奖的教育方面包括一些旨在将本科生融入该系研究生活的举措。这些措施包括开发新的课程和新的本科系列研讨会。在研究生阶段，正在设计一个新的生物化学课程。这包括在化学系设立一个新的学位选择。＊＊＊

Raleigh 9600866 Elucidating how the amino acid sequence determines structure, the protein folding problem, is a central issue in modern structural biology. This proposal describes an integrated approach to studying the folding of the protein L9, a multidomain ribosomal protein. This protein forms an interesting bilobal structure with a compact N-terminal domain connected by a long solvent exposed a-helix to a compact C-terminal domain. The N-terminal domain is a stable folding unit and represents one of the simpler examples of an important class of layered sheet-helix structures. This type of structure is found in other ribosomal proteins and in many small RNA binding domains. Relatively little is known about the interactions which stabilize this important structure or how it folds. The L9 protein also offers a excellent model system to study the folding of multi-domain proteins. In recent years, considerable effort has been devoted to elucidating the folding pathways of single domain globular proteins but much less is known about the folding of multidomain proteins. The folding pathway of this protein will be explored using kinetic measurements. The equilibrium and kinetic folding transitions of the isolated domain and of the full L9 protein will be studied. These experiments will indicate if interactions between domains affect the folding or stability of the protein. The unfolded state will be characterized by Nuclear Magnetic Resonance and Circular Dichroism. Studies of peptide fragments will be used to address the role of local interactions in stabilizing the N-terminal domain. These studies will also identify any potential folding initiation sites. Fragment complementation experiments will be used to probe the role of tertiary interactions in stabilizing nascent secondary structure. Taken together, these experiments will provide a detailed description of the folding pathway and of the interactions which stabilize this interesting protein. One of the major challenges of teaching at a lar ge university is balancing undergraduate education with research. Many of the students feel that they are "lost in the shuffle" or that undergraduate education is ranked a distant third behind research and graduate education. On the other hand, the available research infrastructure has the potential to allow undergraduates unique opportunities. The educational aspects of this Career Award include a number of initiatives designed to integrate undergraduate students into the research life of the department. A new course "Current Trends in Biological Chemistry" is being developed. This course is designed to introduce undergraduates to research areas in modern biological chemistry and is built around the departmental seminar series in biological chemistry. An informal seminar series designed to introduce students to the many career options available to chemists and biochemists is also being developed. One of the core undergraduate courses, physical chemistry laboratory is being extensively revised and redefined. New, more relevant experiments have been introduced and more interactive methods of teaching are being used. At the graduate level, a new program in biological chemistry is being designed. This includes a new seminar series, which also forms the basis of the course "Current Trends in Biological Chemistry': the establishment of a new degree option in the Department of Chemistry, the design of a new course on Protein Structure and the writing of an interdepartmental training grant. %%% Elucidating how the amino acid sequence determines structure, the protein folding problem, is a central issue in modern structural biology. This proposal describes an integrated approach to studying the folding of the multidomain ribosomal protein L9. Considerable effort has been devoted to elucidating the folding pathways of single domain globular proteins but much less is known about the folding of multidomain proteins. The isolated domains of this molecule are also of interest. The N-terminal domain is a st able folding unit and represents one of the simpler examples of an important type of structure found in ribosomal proteins and in small RNA binding domains. One of the major challenges of teaching at a large university is balancing undergraduate education with research. Many of the students feel that they are "lost in the shuffle" and that undergraduate education is ranked a distant third behind research and graduate education. The educational aspects of this Career Award include a number of initiatives designed to integrate undergraduate students into the research life of the department. These include the development of new courses and a new undergraduate seminar series. At the graduate level, a new program in biological chemistry is being designed. This includes the establishment of a new degree option in the Department of Chemistry. ***