SUBMILLISECOND LASER T JUMP INDUCED PROTEIN FOLDING

最近从核磁共振、圆二色谱、荧光光谱 和其他光谱学研究表明，在折叠时，许多小蛋白质 可以在不到一个小时内形成二级和三级结构元件。 毫秒 早期和快速形成的结构可能是 普遍重要的自然和设计的可折叠性 多肽序列。 本研究的长期目标是 通过实验探索这些快速动力学，并将其用作直接测试 分析和计算（如分子动力学）折叠 模型最终，这些实验将提供平均时间图， 蛋白质集合体在折叠过程中的早期空间进化 过程 具体目标是：1）进一步发展一种快速的激光诱导 可探测蛋白质骨架运动温度跳跃装置 在14 ns - 50 ms时间尺度上的重折叠期间。 特别是要 增加了直接检测二级结构形成的能力， 除了我们目前检测三级接触的能力之外 崩溃（collapse）。2)为了衡量中学和中学教育的时间尺度， 具有非常快的动力学阶段的蛋白质三级结构形成 在蛋白质的不同位置。 3)利用这段时间- 解析的结构信息以及能量信息（例如， 反应势垒由温度依赖性决定， 动力学），以比较实验结果与理论和 计算模型预测。 将研究一系列特定系统以采样结构 范围从所有的α基序到大部分的β基序。 我们最初提出的 系统包括脱辅基肌红蛋白、细胞色素c、泛素和 磷酸甘油酸激酶，所有这些都具有已知的亚ms折叠阶段。 这些蛋白质的表达系统对我们来说是可用的， 我们探测特定的三级接触和二级结构的网站- 定向诱变和同位素标记。

It has recently become clear from NMR, circular dichroism, fluorescence and other spectroscopic studies that upon folding, many small proteins can form secondary and tertiary structure elements in less than a millisecond. Early and rapidly formed structure may turn out to be universally important for the foldability of natural and designed polypeptide sequences. It is the long term goal of this research to probe these fast dynamics experimentally and to use them as direct tests of analytical and computational (e.g. molecular dynamics) folding models. Eventually, these experiments will provide average temporal maps of the early spatial evolution of protein ensembles during the folding process. The specific aims are: 1) To further develop a fast laser-induced temperature-jump apparatus which can probe protein backbone motions during refolding on a 14 ns - 50 ms time scale. In particular, we will add the capability of detecting secondary structure formation directly, in addition to our present capability for detecting tertiary contact formation (collapse). 2) To measure the time scales of secondary and tertiary structure formation of proteins with very fast kinetic phases at different locations within the proteins. 3) To use such time- resolved structural information, as well as energetic information (e.g. reaction barriers as determined by the temperature dependence of the kinetics) to compare experimental results with theoretical and computational model predictions. A series of specific systems will be studied to sample structures ranging from all alpha to mostly beta motifs. Our initially proposed systems include apomyoglobin, cytochrome c, ubiquitin, and phosphoglycerate kinase, all of which have known sub-ms folding phases. The expression systems for these proteins are available to us, allowing us to probe specific tertiary contacts and secondary structure by site- directed mutagenesis and isotopic labeling.