Secondary structure and dosage of soluble and membrane proteins by attenuated total reflection Fourier‐transform infrared spectroscopy on hydrated films

Abstract

Attenuated total reflection Fourier‐transform infrared spectroscopy of thin hydrated films of soluble and membrane protein included in a phospholipid bilayer is shown to provide useful information as to the secondary structure of the protein. The analysis of the amide I band of deuterated samples by Fourier self‐deconvolution followed by a curve fitting was performed by a new procedure in which all the input parameters are generated by the computer rather than by the investigator. The results of this analysis provide a correct estimation of the α‐helix and β‐sheet structure content with a standard deviation of 8.6% when X‐ray structures are taken as a reference. We also show that the orientation of the different secondary structures resolved by the Fourier self‐deconvolution/curve‐fitting procedure and of the phospholipid acyl chains can be simultaneously evaluated for membrane proteins reconstituted in a lipid bilayer. Of special interest for reconstitution of membrane proteins, the lipid/protein ratio can be accurately and quickly determined from the infrared spectrum.