NMR APPROACHES TO THE PROTEIN FOLDING PROBLEM

广泛的长期目标是双重的。 一是发展 方法，将扩展的权力，解决核磁共振超出了研究 天然球状蛋白的研究，非天然和非小叶 proteins. 第二个目标是利用核磁共振来了解蛋白质 通过研究部分折叠蛋白质的结构， 并通过评估氨基酸取代对结构的影响， 稳定性和折叠动力学。 这些研究将构成以下方面的基础： 使用核磁共振来了解Gly-X-Y模式中的中断，发现 在胶原蛋白疾病中，如成骨不全和Ehlers Danlos 综合症，可导致严重疾病。 第一个目的是表征的部分折叠状态， 豚鼠α-乳白蛋白，以阐明蛋白质折叠的性质 中间体的 更具体地说，我们希望了解 熔融球状态包含二级结构的区域， 三级相互作用在稳定这些区域中是重要的， 二级结构 我们将通过以下方式来表征部分折叠状态： 1H NMR方法，并通过同位素标记和杂原子2D和3D NMR 实验 关键突变体将被用来评估序列的影响。 分子的二级结构和三级相互作用的变化 折叠状态 第二个目标是首次获得单个残留物， 三螺旋肽的分配和NMR溶液结构， 确定单个氨基酸在稳定三联体中的作用 螺旋并了解关键残基如何指导蛋白质折叠。 我们将 通过1D NMR检查（Gly-X-Y）序列变化对 形成的三股螺旋的量，以及 折页. 为了获得溶液结构，我们将首先设计合成 三螺旋肽，以促进自旋系统的鉴定 过程 然后，我们提出了1H NMR实验，以及杂波NMR实验， 这些实验可以让我们进行序列共振 分配，并区分链间和链内NOE。

The broad long term objectives are twofold. One is to develop methods that will extend the power of solution NMR beyond the study of native globular proteins to the study of nonnative and nonglobular proteins. The second objective is to use NMR to understand protein folding mechanisms by studying structures of partially folded proteins, and by assessing the effect of amino acid substitutions on structure, stability and folding kinetics. These studies will form the basis for using NMR to understand how interruptions in the Gly-X-Y pattern, found in collagen diseases like Osteogenesis Imperfecta and Ehlers Danlos Syndrome, can result in serious disease. The first aim is to characterize the partially folded state of guinea pig alpha-lactalbumin to elucidate the nature of protein folding intermediates. More specifically, we wish to learn which regions of the molten globule state contain regions of secondary structure and whether tertiary interactions are important in stabilizing these regions of secondary structure. We will characterize the partially folded state by 1H NMR methods, and by isotope labelling and heteronuclear 2D and 3D NMR experiments. Key mutants will be made to assess the effects of sequence change on secondary structure and tertiary interactions of the partially folded state. The second aim is to obtain, for the first time, individual residue assignments and the NMR solution structure of triple helical peptides, to determine the role of individual amino acids in stabilizing the triple helix and to understand how key residues direct protein folding. We will examine the effects, by 1D NMR, of (Gly-X-Y) sequence changes on the amount of triple helix formed, and on the kinetics and thermodynamics of folding. To obtain the solution structure we will first design synthetic triple helical peptides to facilitate the spin system identification process. Then we propose 1H NMR experiments as well as heteronuclear NMR experiments that should allow us to perform sequential resonance assignments, and distinguish inter from intra strand NOE's.