Characterization of Macromolecular Dynamics Using Para-hydrogen Induced Polarization of Nuclear Spins

在这个由化学系化学结构、动力学和机制A(CSDM-A)计划资助的项目中，德克萨斯农工大学的克里斯蒂安·希尔蒂教授和一组研究生正在使用核磁共振波谱(核磁共振，医学技术中使用的磁共振成像的表亲)来研究蛋白质如何折叠以获得其三维结构。蛋白质的结构、结构变化和分子相互作用决定了它们在最基本的生命过程中的功能。虽然即使在这些大分子中也有可能确定原子的三维排列，但测量往往太慢，无法解决随时间发生的变化。通常嵌入细胞膜的蛋白质构成了额外的挑战，因为它们依赖于与脂分子的相互作用，即使在纯化进行研究时也是如此。在这个项目中，研究人员正在通过使用一种名为核自旋的对氢诱导极化的方法来克服其中的一些挑战，这种方法增强了核磁共振的信号，使更快地实现这些测量成为可能。随着这些方法的应用，该项目有助于深入了解膜蛋白折叠这一长期存在的问题，尽管已了解相关分子的折叠三维结构，但该问题的许多方面仍然未知。参与这一研究项目的研究生正在获得对他们未来的职业生涯有价值的知识和技能，无论是基础化学研究还是核磁共振在化学分析、医学和其他科学技术领域的应用。该项目开发了表征非平衡大分子过程中结构变化的方法，目的是阐明β-桶状膜蛋白的折叠和插入机制。核磁共振已被证明能够为结构和分子动力学的确定提供化学选择性。核磁共振波谱通过对氢致极化超极化的使用，使得在非平衡条件下的这些结构的阐明是兼容的。富含在准自旋态的氢被输送到生物分子样品中，在原位产生小分子的超极化。第一个目的是证明使用这种对氢增强来表征与模式蛋白质二氢叶酸还原酶的分子间相互作用。发展了多维蛋白质核磁共振实验，利用了再生和向蛋白质传递超极化的能力，并能够测量结合界面结构。在第二个目的中，设计了用于对氢极化的催化剂，其具体地与应用于生物大分子的样品相兼容。最后，在第三个目的中，将这种超极化分子的核磁共振波谱应用于β-桶蛋白OmpX和OmpA的膜插入过程的结构和动力学表征，以及它们与伴侣蛋白的相互作用。AIMS 1和AIMS 2中开发的方法提供了在相关时间尺度上跟踪这些大分子过程所需的分辨率。除了前面提到的物理、生物物理化学和核磁共振S技术方面的学生培训外，该研究项目的教育部分还包括为本科实验室开发低场核磁共振仪器(以及最终的公开演示)，以及为K-12学生和教师开发以酶催化为主题的实验试剂盒(“ChemBox”)。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

In this project funded by the Chemical Structure, Dynamics and Mechanisms A (CSDM-A) program of the Chemistry Division, Professor Christian Hilty of Texas A&M University and a team of graduate students are using nuclear magnetic resonance spectroscopy (NMR, a cousin of the magnetic resonance imaging used in medical technology) to investigate how proteins fold to attain their three-dimensional structure. The structure, structural changes and molecular interactions of proteins are responsible for their function in the most basic processes of life. While it is possible to determine the three-dimensional arrangement of atoms even in these large molecules, the measurements often are too slow to resolve changes that occur over time. Proteins normally embedded in the cell membrane pose additional challenges, because they depend on the interactions with lipid molecules even when they are purified for study. In this project, the investigators are overcoming some of these challenges by using a method called para-hydrogen induced polarization of nuclear spins, which enhances the signals of nuclear magnetic resonance and makes it possible to achieve these measurements more rapidly. With the application of these methods, the project contributes insights into the longstanding problem of membrane protein folding, of which many aspects remain unknown despite the knowledge of the folded, three-dimensional structures of the molecules involved. The graduate students involved in this research project are gaining knowledge and skills that will be valuable in their future career pursuits, be these in fundamental chemistry research or in the applications of NMR in chemical analysis, medicine, and other areas of science and technology.The project develops methods for the characterization of structural changes in non-equilibrium macromolecular processes, motivated by the goal to elucidate the mechanisms of folding and insertion of beta-barrel membrane proteins. The proven ability of NMR to provide chemical selectivity for the determination of structure and molecular dynamics is exploited. NMR spectroscopy is made compatible with the elucidation of these structures under non-equilibrium conditions through the use of hyperpolarization by para-hydrogen induced polarization. Hydrogen enriched in the para-spin state is delivered to biomolecular samples, to generate hyperpolarization of small molecules in-situ. A first aim demonstrates the use of this para-hydrogen enhancement for characterizing intermolecular interactions with the model protein dihydrofolate reductase. Multi-dimensional protein NMR experiments are developed that exploit the ability to regenerate and transfer hyperpolarization to the protein, and that enable measurement of the binding interface structure. In a second aim, catalysts for para-hydrogen polarization are designed, which are specifically compatible with the application to samples of biological macromolecules. Finally, in a third aim, NMR spectroscopy of thus hyperpolarized molecules is applied to the structural and kinetic characterization of the membrane insertion process of the beta-barrel proteins OmpX and OmpA, and of their interaction with chaperones. The methods developed in aims 1 and 2 provide the resolution necessary to follow these macromolecular processes on relevant time scales. In addition to the aforementioned student training in physical and biophysical chemistry and nuclear magnetic resonance s techniques, the educational component of this research project includes the development of low-field NMR instrumentation for undergraduate laboratories (and eventual public demonstrations), and enzyme catalysis themed experimental kits ("ChemBoxes") for K-12 students and teachers.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.