Rich Peter R, Iwaki Masayo
Glynn Laboratory of Bioenergetics, Department of Biology, University College London, Gower Street, London, U.K.
Mol Biosyst. 2007 Jun;3(6):398-407. doi: 10.1039/b702328f. Epub 2007 Apr 30.
We describe techniques that can be used in conjunction with modern attenuated total reflection (ATR) infrared micro-prisms to allow proteins to be manipulated cyclically between different states whilst simultaneously monitoring both mid-IR and UV/visible/near IR changes. These methods provide increased flexibility of the types of changes that can be induced in proteins in comparison to transmission methods. Quantitative measurements can be made of vibrational changes associated with conversion between stable catalytic reaction intermediates, ligand binding and oxidation-reduction. Both hydrophobic and soluble proteins can be analysed and the ability to induce transitions repetitively allows IR difference spectra to be acquired at a signal/noise sufficient to resolve changes due to specific cofactors or amino acids. Such spectra can often be interpreted at the atomic level by standard IR methods of comparisons with model compounds, by isotope and mutation effects and, increasingly, by ab initio simulations. Combination of such analyses with atomic 3D structural models derived from X-ray and NMR studies can lead to a deeper understanding of molecular mechanisms of enzymatic reactions.
我们描述了一些技术，这些技术可与现代衰减全反射（ATR）红外微棱镜结合使用，使蛋白质能够在不同状态之间循环操作，同时监测中红外和紫外/可见/近红外的变化。与透射方法相比，这些方法增加了蛋白质中可诱导变化类型的灵活性。可以对与稳定催化反应中间体之间的转化、配体结合和氧化还原相关的振动变化进行定量测量。疏水性蛋白质和可溶性蛋白质都可以进行分析，并且重复诱导转变的能力使得能够以足以分辨特定辅因子或氨基酸引起的变化的信噪比获取红外差光谱。通过与模型化合物进行比较的标准红外方法、同位素和突变效应，以及越来越多地通过从头算模拟，此类光谱通常可以在原子水平上进行解释。将此类分析与源自X射线和核磁共振研究的原子三维结构模型相结合，可以更深入地理解酶促反应的分子机制。
