利用x射线自由电子激光进行结构酶学研究

IF 2.3 2区物理与天体物理 Q3 CHEMISTRY, PHYSICAL

Structural Dynamics-Us Pub Date : 2016-12-15 DOI:10.1063/1.4972069

Christopher Kupitz, J. L. Olmos, M. Holl, L. Tremblay, K. Pande, S. Pandey, D. Oberthür, M. Hunter, M. Liang, A. Aquila, J. Tenboer, George D. Calvey, Andrea M. Katz, Yujie Chen, M. Wiedorn, J. Knoška, A. Meents, Valerio Majriani, Tyler Norwood, I. Poudyal, T. Grant, Mitchell D. Miller, Weijun Xu, A. Tolstikova, A. Morgan, M. Metz, J. Martin-Garcia, James D. Zook, Shatabdi Roy-Chowdhury, J. Coe, Nirupa Nagaratnam, Domingo Meza, R. Fromme, S. Basu, M. Frank, T. White, A. Barty, S. Bajt, O. Yefanov, H. Chapman, N. Zatsepin, G. Nelson, U. Weierstall, J. Spence, P. Schwander, L. Pollack, P. Fromme, A. Ourmazd, G. Phillips, Marius Schmidt

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引用次数: 98

摘要

混合-注射系列晶体学(MISC)是一种用于成像酶催化反应的技术，在这种反应中，小蛋白质晶体在被x射线脉冲探测之前与底物混合。与流式细胞研究相比，这种方法有几个优点。它提供了(i)室温下接近原子分辨率的结构，(ii)从微秒到秒的时间分辨率，以及(iii)方便的反应引发。它通过使用飞秒x射线脉冲来避免辐射损伤，从而将损伤和化学物质分离开来。在这里，我们通过研究结核分枝杆菌ß-内酰胺酶微晶与头孢曲松抗生素溶液的反应，证明了MISC在x射线自由电子激光下是可行的。载脂蛋白ß-内酰胺酶和头孢曲松结合形式的电子密度图分别在2.8 Å和2.4 Å分辨率下得到。这些结果为研究循环和非循环反应铺平了道路，并代表了许多底物触发生物反应的时间分辨结构动力学的新领域。

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Structural enzymology using X-ray free electron lasers

Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.

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来源期刊

Structural Dynamics-Us CHEMISTRY, PHYSICALPHYSICS, ATOMIC, MOLECU-PHYSICS, ATOMIC, MOLECULAR & CHEMICAL

CiteScore

5.50

自引率

3.60%

发文量

审稿时长

16 weeks

期刊介绍： Structural Dynamics focuses on the recent developments in experimental and theoretical methods and techniques that allow a visualization of the electronic and geometric structural changes in real time of chemical, biological, and condensed-matter systems. The community of scientists and engineers working on structural dynamics in such diverse systems often use similar instrumentation and methods. The journal welcomes articles dealing with fundamental problems of electronic and structural dynamics that are tackled by new methods, such as: Time-resolved X-ray and electron diffraction and scattering, Coherent diffractive imaging, Time-resolved X-ray spectroscopies (absorption, emission, resonant inelastic scattering, etc.), Time-resolved electron energy loss spectroscopy (EELS) and electron microscopy, Time-resolved photoelectron spectroscopies (UPS, XPS, ARPES, etc.), Multidimensional spectroscopies in the infrared, the visible and the ultraviolet, Nonlinear spectroscopies in the VUV, the soft and the hard X-ray domains, Theory and computational methods and algorithms for the analysis and description of structuraldynamics and their associated experimental signals. These new methods are enabled by new instrumentation, such as: X-ray free electron lasers, which provide flux, coherence, and time resolution, New sources of ultrashort electron pulses, New sources of ultrashort vacuum ultraviolet (VUV) to hard X-ray pulses, such as high-harmonic generation (HHG) sources or plasma-based sources, New sources of ultrashort infrared and terahertz (THz) radiation, New detectors for X-rays and electrons, New sample handling and delivery schemes, New computational capabilities.