GROEL-MEDIATED PROTEIN FOLDING

描述：该提案是早期资助的一项提案的延续。 该提案旨在进一步界定GroEL的作用及其 Cochaperonin，GroES，在体内蛋白质折叠中的作用。 未折叠多肽形式 与GroEL/GroEs的三元复合物，并结合反式，即，上 与GroES相反侧，或顺式结合，即，与Groes站在同一阵线 在由GroEL和GroES形成的中央腔室中。 折叠可以进行 在反式和顺式侧不同。 一个主要目标（Aim 1）是 精确地确定了E.杆菌蛋白 它们是GroEL的底物。 这些研究将利用快速发病 在P.I.中构建GroEL的温度敏感突变体。的 实验室，以解决各种问题：大蛋白质（>60 kDa） 由于它们的大小，它们也不能结合顺式 GroEL？ 如果是这样，它们的错误折叠形式是否有效地结合在 transs，即它们是否被GroEL展开并释放以用于下一轮 在溶液中折叠？ 是小蛋白质（<10 kDa），由于 它们的大小可能比它们结合GroEL更快地折叠， GroEL/GroES？ 如果是这样，私家侦探。将利用这些蛋白质 通过氘交换和NMR光谱进行机理研究， 顺式介导的折叠 第二个目标（目标2）是直接观察 与底物结合GroEL相关的构象变化。 蛋白酶 和色氨酸荧光研究提出表征 伴随亚稳态rubisco结合的构象变化， 线粒体苹果酸脱氢酶（mMDH）形成GroEL。 停流 芘标记的Rubisco和二氢叶酸的芘荧光研究 还原酶（DHFR）的作用。 后一项研究将允许 展开动力学的定量比较（通过荧光监测 变化）与结合动力学（通过荧光各向异性监测 测量），并帮助确定“类似本地”的中间产品是否需要 首先展开，以便绑定GroEL或是否可以绑定GroEL，然后 展开 第三个目标（目标3）是确定底物的命运 分子起始于具有GroEL/GroES的顺式三元复合物。 可以 例如，多肽使顺式侧处于非天然状态，或者是 它们只在原生状态下释放， 仅从反侧发生（如某些电流假设的那样 模型）？ 在P.I.的 实验室，结合GroEL突变体“陷阱”， 结合但不释放非天然多肽，将用于测试 这些假设。

DESCRIPTION: This proposal, which is a continuation of an earlier funded proposal, is aimed at further defining the role of GroEL and its cochaperonin, GroES, in protein folding in vivo. Unfolded polypeptides form ternary complexes with GroEL/GroEs and bind either trans, i.e., on the opposite side from GroES, or bind cis, i.e., on the same side as GroES within a central chamber formed from GroEL and GroES. Folding may proceed differently at the trans and cis sides. A major goal (Aim 1) is to determine precisely the perecentage and identity of the E. coli proteins which are substrates of GroEL. These studies will exploit rapid onset temperature-sensitive mutants of GroEL to be constructed in the P.I.'s laboratory, to address a variety of questions: Are large proteins (>60 kDa) which should be incapable of binding cis because of their size also substrates of GroEL? if so, do their misfolded forms bind productively in trans, i.e, are they unfolded and released by GroEL for a subsequent round of folding in solution? Are small proteins (<10 kDa), which because of their size potentially can fold more rapidly than they can bind GroEL, also substrates of GroEL/GroES? If so, the P.I. will use such proteins for mechanistic studies by deuterium exchange and NMR spectroscopy to elucidate cis-mediated folding. A second goal (Aim 2) is to observe directly the conformation changes associated with substrate binding to GroEL. Protease and tryptophan fluorescence studies are proposed to characterize conformational changes which accompany the binding of metastable rubisco and mitochondrial malate dehydrogenase (mMDH) forms to GroEL. Stopped-flow pyrene fluorescence studies of pyrene-labeled rubisco and dihydrofolate reductase (DHFR) are also proposed. The latter studies would permit quantitative comparisons of unfolding kinetics (monitored by fluorescence changes) with binding kinetics (monitored by fluorescence anisotropy measurements) and help establish whether "native-like" intermediates need to first unfold in order to bind GroEL or whether they can bind GroEL and then unfold. A third goal (Aim 3) is to determine the fate of substrate molecules starting in a cis ternary complex with GroEL/GroES. Can polypeptides, for example, leave the cis side in a non-native state, or are they released only in the native state with release of non-native forms occuring only from the trans side (as is hypothesized by some current models)? A "cis-only" chaperonin complex constructed in the P.I.'s laboratory, in conjunction with GroEL-mutant "traps" that are capable of binding but not releasing non-native polypeptides, will be used to test these hypotheses.