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


Correspondence to E. Goormaghtigh, Université Libre de Bruxelles, Campus Plaine CP 206/2, B-1050 Bruxelles, Belgium


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.