Klink Björn U, Scheidig Axel J
Department of Biophysics, Division of Structural Biology, Saarland University, Homburg/Saar, Germany.
BMC Struct Biol. 2010 Oct 25;10:38. doi: 10.1186/1472-6807-10-38.
In kinetic crystallography, the usually static method of X-ray diffraction is expanded to allow time-resolved analysis of conformational rearrangements in protein structures. To achieve this, reactions have to be triggered within the protein crystals of interest, and optical spectroscopy can be used to monitor the reaction state. For this approach, a modified form of H-Ras p21 was designed which allows reaction initiation and fluorescence readout of the initiated GTPase reaction within the crystalline state. Rearrangements within the crystallized protein due to the progressing reaction and associated heterogeneity in the protein conformations have to be considered in the subsequent refinement processes.
X-ray diffraction experiments on H-Ras p21 in different states along the reaction pathway provide detailed information about the kinetics and mechanism of the GTPase reaction. In addition, a very high data quality of up to 1.0 Å resolution allowed distinguishing two discrete subconformations of H-Ras p21, expanding the knowledge about the intrinsic flexibility of Ras-like proteins, which is important for their function. In a complex of H-Ras•GppNHp (guanosine-5'-(β,γ-imido)-triphosphate), a second Mg2+ ion was found to be coordinated to the γ-phosphate group of GppNHp, which positions the hydrolytically active water molecule very close to the attacked γ-phosphorous atom.
For the structural analysis of very high-resolution data we have used a new 'two-chain-isotropic-refinement' strategy. This refinement provides an alternative and easy to interpret strategy to reflect the conformational variability within crystal structures of biological macromolecules. The presented fluorescent form of H-Ras p21 will be advantageous for fluorescence studies on H-Ras p21 in which the use of fluorescent nucleotides is not feasible.
在动力学晶体学中，通常静态的X射线衍射方法得到扩展，以允许对蛋白质结构中的构象重排进行时间分辨分析。为实现这一点，必须在感兴趣的蛋白质晶体内部引发反应，并且可以使用光学光谱来监测反应状态。对于这种方法，设计了一种修饰形式的H-Ras p21，它能够在晶体状态下引发反应并对引发的GTP酶反应进行荧光读出。在后续的精修过程中，必须考虑由于反应进行导致的结晶蛋白质内部的重排以及蛋白质构象中相关的异质性。
对反应途径中不同状态的H-Ras p21进行的X射线衍射实验提供了有关GTP酶反应动力学和机制的详细信息。此外，高达1.0 Å分辨率的非常高的数据质量使得能够区分H-Ras p21的两种离散亚构象，扩展了对Ras样蛋白固有灵活性的认识，这对其功能很重要。在H-Ras•GppNHp（鸟苷-5'-(β,γ-亚氨基)-三磷酸）复合物中，发现第二个Mg2+离子与GppNHp的γ-磷酸基团配位，这使得水解活性水分子非常靠近被攻击的γ-磷原子。
对于超高分辨率数据的结构分析，我们使用了一种新的“双链各向同性精修”策略。这种精修提供了一种替代且易于解释的策略，以反映生物大分子晶体结构内的构象变异性。所呈现的荧光形式的H-Ras p21将有利于对H-Ras p21进行荧光研究，在这些研究中使用荧光核苷酸是不可行的。
