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，可能会有危险。正确的折叠和错误的折叠是所有细胞功能和保持健康的关键。然而，关于这一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，通过离散的、明确定义的中间体在离散的路径中折叠，还是像所谓的新观点那样，它们通过多个独立的、无关的路径折叠。这项工作使用了在上一次获奖中开发的一种强大的新方法来理解蛋白质折叠问题，并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、本科生和高中生积极参与现代结构生物学前沿的实践研究。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众多生物学功能的生物物理性质非常敏感。因此，它能够对蛋白质科学研究做出重要贡献，特别是当用氨基酸分辨率的核磁共振测量时。然而，核磁共振仅限于研究高浓度下相对较小的蛋白质数量。PI实验室最近的工作开发了一种先进的质谱分析(HX MS)，可以使用微量的生物材料在以前无法接触到的大型复杂蛋白质系统中以接近氨基酸的分辨率测量HX。这项技术将被用于研究蛋白质折叠问题，这是生命科学中许多重要问题的基础。蛋白质折叠本身的详细机制还不清楚，主要是因为瞬时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 MS技术能够跟踪动力学折叠过程，并描述将最初未折叠的蛋白质带到最终自然状态的中间形式和途径。在这项拟议的工作中，HX MS技术将被用于研究蛋白质如何折叠，为什么它们以这种方式折叠，以及辅助伴侣分子如何发挥促进折叠的作用。如何解决这个问题将在生物上典型的大蛋白质的研究中解决，不同于几乎所有以前的研究(无论是理论上的还是实验的)，这些研究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以高结构分辨率研究实验室设计的蛋白质的折叠来解决，这些蛋白质还没有通过生物进化来塑造和选择。伴侣功能将通过研究蛋白质的详细折叠来解决，而蛋白质包含在活性的GroEL伴侣素腔中。这项工作可能会很好地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，这是以前根本不可能的。这项工作由生物学系分子和细胞生物科学部的分子生物物理组资助，并由MPS化学部的生命过程化学计划共同资助。

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.