Tatulian Suren A
Department of Physics, University of Central Florida, Orlando, FL, USA.
Methods Mol Biol. 2013;974:177-218. doi: 10.1007/978-1-62703-275-9_9.
Fourier transform infrared (FTIR) spectroscopy is widely used in structural characterization of proteins or peptides. While the method does not have the capability of providing the precise, atomic-resolution molecular structure, it is exquisitely sensitive to conformational changes occurring in proteins upon functional transitions or upon intermolecular interactions. Sensitivity of vibrational frequencies to atomic masses has led to development of "isotope-edited" FTIR spectroscopy, where structural effects in two proteins, one unlabeled and the other labeled with a heavier stable isotope, such as (13)C, are resolved simultaneously based on spectral downshift (separation) of the amide I band of the labeled protein. The same isotope effect is used to identify site-specific conformational changes in proteins by site-directed or segmental isotope labeling. Negligible light scattering in the infrared region provides an opportunity to study intermolecular interactions between large protein complexes, interactions of proteins and peptides with lipid vesicles, or protein-nucleic acid interactions without light scattering problems often encountered in ultraviolet spectroscopy. Attenuated total reflection FTIR (ATR-FTIR) is a surface-sensitive version of infrared spectroscopy that has proved useful in studying membrane proteins and lipids, protein-membrane interactions, mechanisms of interfacial enzymes, and molecular architecture of membrane pore or channel forming proteins and peptides. The purpose of this article was to provide a practical guide to analyze protein structure and protein-membrane interactions by FTIR and ATR-FTIR techniques, including procedures of sample preparation, measurements, and data analysis. Basic background information on FTIR spectroscopy, as well as some relatively new developments in structural and functional characterization of proteins and peptides in lipid membranes, are also presented.
傅里叶变换红外（FTIR）光谱法在蛋白质或肽的结构表征中被广泛应用。虽然该方法无法提供精确的原子分辨率分子结构，但它对蛋白质在功能转变或分子间相互作用时发生的构象变化极为敏感。振动频率对原子质量的敏感性促使了“同位素编辑”FTIR光谱法的发展，在这种方法中，基于标记蛋白质酰胺I带的光谱下移（分离），可同时解析两种蛋白质中的结构效应，一种未标记，另一种用较重的稳定同位素如（13）C标记。相同的同位素效应可通过定点或片段同位素标记来识别蛋白质中位点特异性的构象变化。红外区域可忽略不计的光散射为研究大型蛋白质复合物之间的分子间相互作用、蛋白质和肽与脂质囊泡的相互作用或蛋白质 - 核酸相互作用提供了机会，而不会出现紫外光谱中经常遇到的光散射问题。衰减全反射FTIR（ATR - FTIR）是一种对表面敏感的红外光谱法，已被证明在研究膜蛋白和脂质、蛋白质 - 膜相互作用、界面酶的机制以及形成膜孔或通道的蛋白质和肽的分子结构方面很有用。本文的目的是提供一份实用指南，介绍如何通过FTIR和ATR - FTIR技术分析蛋白质结构和蛋白质 - 膜相互作用，包括样品制备、测量和数据分析的程序。还介绍了FTIR光谱法的基本背景信息，以及脂质膜中蛋白质和肽的结构与功能表征方面的一些相对较新的进展。
