SUBMILLISECOND LASER T JUMP INDUCED PROTEIN FOLDING

最近从核磁共振、圆二色谱、荧光 和其他光谱研究表明，在折叠过程中，许多小蛋白 可以在不到一年的时间内形成二级和三级结构元素 毫秒。早期和快速形成的结构可能被证明是 对自然和设计的可折叠性具有普遍重要性 多肽序列。这项研究的长期目标是 通过实验来探测这些快速动力学，并将其用作直接测试 分析和计算(例如分子动力学)折叠 模特们。最终，这些实验将提供平均的时间地图 折叠过程中蛋白质系综的早期空间进化 进程。 具体目标是：1)进一步开发快速激光诱导 可探测蛋白质骨架运动的温度跳跃仪 在14 ns-50毫秒的时间尺度上进行重折叠。特别是，我们将 增加了直接检测二级结构形成的能力， 除了我们目前检测第三次接触的能力之外 队形(崩塌)。2)测量次要和次要的时间尺度 具有极快动力学相的蛋白质的三级结构形成 在蛋白质中的不同位置。3)利用这些时间- 解析的结构信息以及能量信息(例如 反应势垒是由温度依赖性决定的 动力学)将实验结果与理论和 计算模型预测。 将对一系列特定的系统进行研究，以抽样结构 从所有的阿尔法主题到大部分的贝塔主题。我们最初提出的 系统包括去肌红蛋白、细胞色素c、泛素和 磷酸甘油酸激酶，所有这些蛋白都有已知的亚ms折叠相。 这些蛋白质的表达系统对我们是可用的，允许 美国将按地点调查特定的第三级接触和二级结构- 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.