Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA.
Proc Natl Acad Sci U S A. 2010 Apr 20;107(16):7281-6. doi: 10.1073/pnas.1002951107. Epub 2010 Apr 6.
We have developed a time-resolved x-ray scattering diffractometer capable of probing structural dynamics of proteins in solution with 100-ps time resolution. This diffractometer, developed on the ID14B BioCARS (Consortium for Advanced Radiation Sources) beamline at the Advanced Photon Source, records x-ray scattering snapshots over a broad range of q spanning 0.02-2.5 A(-1), thereby providing simultaneous coverage of the small-angle x-ray scattering (SAXS) and wide-angle x-ray scattering (WAXS) regions. To demonstrate its capabilities, we have tracked structural changes in myoglobin as it undergoes a photolysis-induced transition from its carbon monoxy form (MbCO) to its deoxy form (Mb). Though the differences between the MbCO and Mb crystal structures are small (rmsd < 0.2 A), time-resolved x-ray scattering differences recorded over 8 decades of time from 100 ps to 10 ms are rich in structure, illustrating the sensitivity of this technique. A strong, negative-going feature in the SAXS region appears promptly and corresponds to a sudden > 22 A(3) volume expansion of the protein. The ensuing conformational relaxation causes the protein to contract to a volume approximately 2 A(3) larger than MbCO within approximately 10 ns. On the timescale for CO escape from the primary docking site, another change in the SAXS/WAXS fingerprint appears, demonstrating sensitivity to the location of the dissociated CO. Global analysis of the SAXS/WAXS patterns recovered time-independent scattering fingerprints for four intermediate states of Mb. These SAXS/WAXS fingerprints provide stringent constraints for putative models of conformational states and structural transitions between them.
我们开发了一种时间分辨 X 射线散射衍射仪，能够以 100-ps 的时间分辨率探测溶液中蛋白质的结构动力学。该衍射仪是在高级光子源的 ID14B BioCARS（先进辐射源联合中心）光束线上开发的，可记录跨越 0.02-2.5 A(-1) 的广泛 q 值的 X 射线散射快照，从而同时覆盖小角 X 射线散射 (SAXS) 和广角 X 射线散射 (WAXS) 区域。为了展示其功能，我们跟踪了肌红蛋白在光解诱导从一氧化碳形式 (MbCO) 到脱氧形式 (Mb) 的转变过程中的结构变化。尽管 MbCO 和 Mb 晶体结构之间的差异很小（rmsd < 0.2 A），但从 100 ps 到 10 ms 记录的超过 8 个数量级的时间分辨 X 射线散射差异在结构上非常丰富，说明了该技术的灵敏度。SAXS 区域中出现的一个强烈的负向特征很快出现，对应于蛋白质体积的突然>22 A(3)膨胀。随后的构象松弛导致蛋白质在大约 10 ns 内收缩到大约比 MbCO 大 2 A(3)的体积。在 CO 从主要停泊位点逸出的时间尺度上，SAXS/WAXS 指纹出现了另一个变化，表明对分离的 CO 位置敏感。对 SAXS/WAXS 模式的全局分析恢复了 Mb 的四个中间状态的时间独立散射指纹。这些 SAXS/WAXS 指纹为构象状态和它们之间的结构转变的假设模型提供了严格的约束。
