NMR STUDIES OF TRIPLE HELICAL PEPTIDES

宏大的长期目标有两个。一是发展 将把溶液核磁共振的能力扩展到研究之外的方法 天然球状蛋白在非天然和非小叶的研究中的应用 蛋白质。第二个目标是使用核磁共振来理解蛋白质。 折叠机制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。这些研究将形成以下基础 使用核磁共振来理解Gly-X-Y模式中的中断是如何发现的 在成骨不全和埃勒斯·丹洛斯等胶原疾病中 综合症，会导致严重的疾病。 第一个目标是刻画部分折叠的状态 豚鼠α-乳清蛋白阐明蛋白质折叠的性质 中间体。更具体地说，我们希望了解 熔融球状状态包含二级结构区域以及是否 第三级相互作用对稳定这些区域非常重要 二级结构。我们将通过以下方式来刻画部分折叠状态 ~1H核磁共振方法，同位素标记和异核2D和3D核磁共振 实验。关键的突变将被用来评估序列的影响 部分分子的二级结构和三级相互作用的变化 折叠状态。 第二个目标是第一次获得个体残留物 三螺旋多肽的归属和核磁共振溶液结构 确定单个氨基酸在稳定三重结构中的作用 并了解关键残基如何指导蛋白质折叠。我们会 用一维核磁共振技术研究(Gly-X-Y)序列变化对蛋白质合成的影响 三螺旋的生成量，以及对其动力学和热力学的影响 折叠。为了得到溶液结构，我们首先要设计合成的 三螺旋多肽有助于自旋系统的鉴定 进程。然后，我们提出了核磁共振氢谱实验和异核核磁共振实验 应该允许我们进行顺序共振的实验 分配，并区分INTER和INTERT 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.