Protein folding: mechanism and principles

蛋白质折叠可能是所有蛋白质功能中最基本的。蛋白质折叠是一个困难的过程，当它出错时是潜在的危险。正确折叠和错误折叠是所有细胞功能和维持健康的关键。然而，对于这个基本过程是如何工作的，仍然没有普遍的共识：蛋白质是像经典观点那样，在离散的途径中通过离散的、定义良好的中间体折叠，还是像所谓的新观点那样，通过多个独立的、不相关的途径折叠。这项工作使用了在上一个奖项中开发的强大的新方法来理解蛋白质折叠问题，并且可能提供有洞察力的信息来详细定义折叠过程并回答该领域的关键问题。这些研究将参与并积极培训、教授和支持博士后、本科生和高中生在现代结构生物学前沿的积极实践研究。自然发生的蛋白质氢交换（HX）行为对允许蛋白质分子完成其无数生物功能的生物物理特性非常敏感。因此，它能够为蛋白质科学研究做出重要贡献，特别是当用核磁共振在氨基酸分辨率上进行测量时。然而，核磁共振仅限于研究相对较小的蛋白质在高浓度下的数量。PI实验室最近的工作开发了一种先进的质谱分析（HX MS），可以在大型复杂蛋白质系统中以接近氨基酸的分辨率测量HX，在以前使用微量生物材料无法达到的情况下。该技术将用于研究蛋白质折叠问题，这是生物科学中许多重要问题的基础。蛋白质折叠本身的详细机制尚不清楚，主要是因为瞬态折叠中间体和折叠途径超出了通常的高分辨率结构方法的范围。最新先进的HX质谱技术能够跟踪动态折叠过程，并描述将最初未折叠的蛋白质携带到最终天然状态的中间形式和途径。在提出的工作中，HX质谱技术将用于询问蛋白质如何折叠，为什么它们以这种方式折叠，以及辅助伴侣分子如何起作用以促进折叠。如何解决的问题将在生物学上典型的大蛋白质的研究中得到解决，而不像几乎所有以前的研究（理论和实验）都是针对只占生物蛋白质组的百分之几的小蛋白质。为什么这个问题将通过在高结构分辨率下研究实验室设计的蛋白质的折叠来解决，这些蛋白质没有通过生物进化形成和选择。伴侣蛋白的功能将通过研究包含在活性GroEL伴侣蛋白腔内的蛋白质的详细折叠来解决。这项工作可能会改变目前的蛋白质折叠模式，更广泛地说，将为蛋白质结构-功能研究提供一个强大的新工具，这在以前是不可能的。这项工作由生物学部分子和细胞生物科学部的分子生物物理集群资助，并由MPS化学学部的生命过程化学项目共同资助。

Protein folding is perhaps the most fundamental of all protein functions. Protein folding is a difficult process and is potentially dangerous when it goes wrong. Correct folding and misfolding are key to the function of all cells and to maintain health. Yet there is still no general agreement about how this basic process works: do proteins fold through discrete well defined intermediates in discrete pathways, as in the classical view, or do they fold by way of multiple independent unrelated pathways as in the so-called new view. This work uses a powerful new approach developed in the last award to understand the protein folding problem, and is likely to provide the insightful information to define the folding process in some detail and answer key questions in the field. These studies will engage and actively train, teach, and support postdoctoral, undergraduate, and high school students in active hands-on research at the forefront of modern structural biology. Naturally occurring protein hydrogen exchange (HX) behavior is very sensitive to the biophysical properties that allow protein molecules to accomplish their myriad biological functions. It has therefore been able to contribute importantly to protein science research, especially when measured by NMR at amino acid resolution. However NMR is limited to the study of relatively small proteins in quantity at high concentrations. Recent work in the PI's laboratory has developed an advanced mass spectrometry analysis (HX MS) that can measure HX at near amino acid resolution in large and complex protein systems in previously unreachable situations using minuscule amounts of biological material. This technique will be used to study the protein folding problem, which lies at the base of many important problems in biological science. The detailed mechanism of protein folding itself is not understood, mainly because transient folding intermediates and folding pathways are beyond the reach of the usual high resolution structural methods. The newly advanced HX MS technology is able to track the kinetic folding process and describe the intermediate forms and pathways that carry an initially unfolded protein to its final native state. In the proposed work, HX MS technology will be used to ask how proteins fold, why they fold in that way, and how helper chaperone molecules function to promote folding. The how question will be addressed in studies of a biologically typical large protein, unlike almost all previous studies (both theoretical and experimental) which have been directed at small proteins that account for only a few percent of the biological proteome. The why question will be addressed by studying at high structural resolution the folding of laboratory-designed proteins that have not been shaped and selected through biological evolution. Chaperone function will be addressed by studying the detailed folding of a protein while it is contained within the active GroEL chaperonin cavity. This work may well change the current protein folding paradigm and, more broadly, will provide a powerful new tool for protein structure-function studies that have simply not been possible before. This work is being funded by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division of BIO, and co-funded by the Chemistry of Life Processes Program in the Chemistry Division of MPS.