Secondary structure and dosage of soluble and membrane proteins by attenuated total reflection Fourier-transform infrared spectroscopy on hydrated films.
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 alpha-helix and beta-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.
Goormaghtigh
E
E
Laboratoire de Chimie Physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, Belgium.
Cabiaux
V
V
Ruysschaert
J M
JM
D001330:Electronic Data Processing; D005583:Fourier Analysis; D008565:Membrane Proteins / Q000737:chemistry*; D010743:Phospholipids; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared / Q000379:methods
D008565:Membrane Proteins; D010743:Phospholipids