UNFOLDED STATES AND FOLDING PATHWAYS BY NMR

描述（改编自申请人摘要）：结构研究 蛋白质未折叠状态的动力学研究具有重要意义 因为他们对蛋白质折叠的起始提供了深刻的见解。 在 此外，一些疾病状态来自折叠缺陷， 最近已经显示出特定蛋白质和AN的稳定性 越来越多的关键细胞内蛋白质似乎是解折叠的， 它们的生物功能状态。 该项目的主要目标是 重点是阐明折叠途径和结构 未折叠状态的表征。 有三个主要的具体目标， 代表了一种综合的方法，利用最近开发的核磁共振 方法： (1)异相NMR方法将用于获得定量的 由两个未折叠蛋白质取样的构象的描述， 酸性未折叠的脱辅基肌红蛋白和低盐未折叠的脱辅基质体蓝蛋白， 几乎完全包含一种类型的二级结构， 状态 根据初步结果，未折叠状态是易处理的， 特别是质外体蓝素具有额外的优点， 形式存在于“折叠”条件下，中性pH和环境温度。 温度，使结果高度适用于蛋白质折叠 在体内和体外的过程。 详细研究了骨架结构 这两种蛋白在未折叠状态下的倾向和多肽动力学 蛋白质将被制成，以及在结构上的变性剂， 展开状态。 (2)质外体氰胺的折叠在核磁共振上是缓慢的 时间尺度和MR研究折叠过程中的真实的时间。 早期的快速折叠步骤之后将是多步停止流动 荧光和氢交换脉冲标记。 3)长期目标是扩展展开状态的工作， 与伴侣蛋白相互作用的研究。 展开的相互作用 蛋白质和蛋白肽将与来自E. 卷曲伴侣蛋白DnaJ，其已被证明结合锌， 类似于糖皮质激素受体的DNA结合域。 这 令人惊讶的结果代表了新的信息， 在蛋白质的折叠和组装中发现了一个新的， 重要信息

DESCRIPTION (Adapted from applicant's abstract): Studies of the structure and dynamics of unfolded states of proteins have assumed great importance for the insights they provide into the initiation of protein folding. In addition, a number of disease states from defects in the folding and stability of specific proteins and an have recently been shown to arise increasing number of key intracellular proteins appear to be unfolded in their biologically functional states. The major goals of this project are focused on the elucidation of folding pathways and the structural characterization of unfolded states. There are three major specific aims, representing an integrated approach that utilizes recently-developed NMR methods: (1) Heteronuclear NMR methods will be used to obtain a quantitative description of the conformations sampled by two unfolded proteins, acid-unfolded apomyoglobin and low-salt-unfolded apoplastocyanin, which contain almost purely a single type of secondary structure in the folded state. The unfolded states are tractable according to preliminary results, and apoplastocyanin in particular has the added advantage that the unfolded form is present under "folding" conditions, neutral pH and ambient temperature, making the results highly applicable-to the protein folding process in vivo and in vitro. A detailed study of backbone structural propensities and polypeptide dynamics in the unfolded state of these two proteins will be made, as well as of denaturants on the structure in the unfolded state. (2) The folding of apoplastocyamin is slow on the NMR timescale and an MR study of the folding process in real time is proposed. The early, fast folding steps will be followed by multiple-step stopped-flow fluorescence and hydrogen exchange pulse labeling. 3) A long-term goal is to extend the work on the unfolded state to include studies of interactions with chaperones. The interaction of unfolded proteins and protpetides will be studies with a small domain from the E. coil chaperone protein DnaJ, which has been shown to bind zinc and to resemble the DNA-binding domain of glucocorticoid receptors. This surprising result typifies the new information that is constantly being uncovered in the protein folding and assembly a continual source of new and important information.