IR Spectroscopy for Protein Secondary Structure

Infrared spectroscopy (especially Fourier-transform infrared spectroscopy, FTIR) is a commonly used technique for studying the secondary structure of proteins. This method is based on the vibrational absorption of peptide bond C=O (amide I) and N-H (amide II) in different components of protein secondary structures (such as α-helix, β-fold, β-turn and irregular curl).

 

The Basic Steps and Principles of Determining Protein Secondary Structure by Using Infrared Spectroscopy

1. Samlpe Preparation

(1) The protein sample is mixed with an appropriate solvent to stabilize it under suitable conditions. Commonly used solvents include D2O or heavy water, as the O-H vibrations of regular water can overlap with certain vibrations of the protein.

(2) The sample can be prepared as a thin film or prepared on an appropriate infrared transparent window material.

 

2. Obtain Infrared Spectrum

(1) The infrared spectrum of the sample is measured using an FTIR spectrometer.

(2) Typically, the areas of interest are 1700-1600 cm^-1 (amide I band) and 1550-1500 cm^-1 (amide II band).

 

3. Data Analysis

(1) Based on the specific infrared absorption of known various secondary structure components, methods such as deconvolution or curve fitting can be used to estimate the relative content of various secondary structures in the sample.

(2) For example, the α-helix usually shows absorption at the position of 1650-1658 cm^-1, while the β-fold shows absorption at the position of 1620-1640 cm^-1.

 

4. Results Interpretation

(1) Based on the obtained data, the relative proportions of various secondary structures in the protein sample can be determined.

(2) These data can be compared and validated with data obtained from other experimental methods (such as circular dichroism or nuclear magnetic resonance).

 

FTIR is a powerful and non-invasive technique for studying the secondary structure of proteins, and is particularly suitable for samples that are not easily analyzed by other techniques such as X-ray crystallography or nuclear magnetic resonance.