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中心问题。 最近的事态发展使这一问题特别及时。大规模的基因组测序工作已经导致我们对蛋白质一级序列的知识爆炸。 不幸的是，仍然不可能预测结构， 顺序 原则上，对折叠过程更透彻的理解应该有助于破译连接序列和结构的密码。对折叠过程的详细了解也将有助于合理修饰蛋白质以增强所需的性质或赋予新的功能。 该项目中描述的研究重点是蛋白质折叠中几个问题的实验研究。链拓扑结构是折叠动力学的主要决定因素的假设将通过对两组蛋白质的折叠进行研究来检验。将测量蛋白质L9的C-末端结构域的折叠动力学，并与理论预测进行比较。在完整的蛋白质中，这个结构域的折叠速度似乎比理论预测的要慢得多。将研究分离结构域的折叠，以测试偏差是否由与蛋白质其余部分的相互作用引起或更基本。 RNA酶Hl的N-末端结构域的结构与L9的N-末端结构域（NTL 9）非常相似，并且将该蛋白质的折叠与NTL 9进行比较。将使用NTL 9作为模型系统来检查未折叠状态在蛋白质折叠和稳定性中的作用。NTL 9中的六个酸性残基中的每一个都被突变，并确定稳定性的pH依赖性。 这些测量提供了有关静电的信息 在未折叠状态下的相互作用。 在折叠过程中静电相互作用的形成将进行动力学测量与这组突变体进行研究。这些研究将通过探索NTL 9在折叠过程中疏水核心的发展的实验来补充。 总之，动力学研究将提供NTL 9折叠的详细情况。

0079406RaleighElucidating how the amino acid sequence determines structure, the protein folding problem, is a central issue in modern structural biology. Recent developments have made this problem particularly timely. Massive genome sequencing efforts have lead to an explosion in our knowledge of the primary sequence of proteins. Unfortunately it is still impossible to predict structure from sequence. In principle, a more thorough understanding of the folding process should aid efforts to decipher the code that links sequence and structure. A detailed understanding of the folding process will also aid efforts to rationally modify proteins to enhance desired properties or confer new functions. The research described in this project focuses on experimental investigations of several issues in protein folding. The hypothesis that chain topology is the major determinant of folding kinetics will be examined by conducting studies of the folding of two sets of proteins. The folding kinetics of the C-terminal domain of the protein L9 will be measured and compared to theoretical predictions. In the intact protein, this domain appears to fold much more slowly than predicted by theory. The folding of the isolated domain will be studied in order to test if the deviation is caused by interactions with the rest of the protein or is more fundamental. The structure of the N-terminal domain of RNase HI is very similar to the N-terminal domain of L9 (NTL9) and the folding of this protein will be compared to NTL9. The role of the unfolded state in protein folding and stability will be examined using NTL9 as a model system. Each of the six acidic residues in NTL9 with be mutated and the pH dependence of the stability determined. These measurements provide information about electrostatic interactions in the unfolded state. The formation of electrostatic interactions during the folding process will be studied by conducting kinetic measurements with this set of mutants. These studies will be complimented by experiments that probe the development of the hydrophobic core of NTL9 during folding. Taken together, the kinetic studies will provide a detailed picture of the folding of NTL9.