학술논문
Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy
Document Type
Report
Source
Nature Protocols. March 1, 2015, p382, 15 p.
Subject
Language
English
ISSN
1754-2189
Abstract
INTRODUCTION Overview IR spectroscopy is an excellent method for biological analysis (1). The introduction of computers based on the Michelson interferometer, the application of the fast Fourier transform algorithm (2) [...]
Fourier transform IR (FTIR) spectroscopy is a nondestructive technique for structural characterization of proteins and polypeptides. The IR spectral data of polymers are usually interpreted in terms of the vibrations of a structural repeat. the repeat units in proteins give rise to nine characteristic IR absorption bands (amides A, B and I-VII). Amide I bands (1,700-1,600 [cm.sup.-1]) are the most prominent and sensitive vibrational bands of the protein backbone, and they relate to protein secondary structural components. In this protocol, we have detailed the principles that underlie the determination of protein secondary structure by FTIR spectroscopy, as well as the basic steps involved in protein sample preparation, instrument operation, FTIR spectra collection and spectra analysis in order to estimate protein secondary-structural components in aqueous (both [H.sub.2]O and deuterium oxide ([D.sub.2]O)) solution using algorithms, such as second-derivative, deconvolution and curve fitting. Small amounts of high-purity (>95%) proteins at high concentrations (>3 mg [ml.sup.-1]) are needed in this protocol; typically, the procedure can be completed in 1-2 d.
Fourier transform IR (FTIR) spectroscopy is a nondestructive technique for structural characterization of proteins and polypeptides. The IR spectral data of polymers are usually interpreted in terms of the vibrations of a structural repeat. the repeat units in proteins give rise to nine characteristic IR absorption bands (amides A, B and I-VII). Amide I bands (1,700-1,600 [cm.sup.-1]) are the most prominent and sensitive vibrational bands of the protein backbone, and they relate to protein secondary structural components. In this protocol, we have detailed the principles that underlie the determination of protein secondary structure by FTIR spectroscopy, as well as the basic steps involved in protein sample preparation, instrument operation, FTIR spectra collection and spectra analysis in order to estimate protein secondary-structural components in aqueous (both [H.sub.2]O and deuterium oxide ([D.sub.2]O)) solution using algorithms, such as second-derivative, deconvolution and curve fitting. Small amounts of high-purity (>95%) proteins at high concentrations (>3 mg [ml.sup.-1]) are needed in this protocol; typically, the procedure can be completed in 1-2 d.