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

TLDR

Attenuated total reflection Fourier‑transform infrared spectroscopy of thin hydrated films of soluble and membrane proteins in a phospholipid bilayer provides useful information on protein secondary structure. The amide I band of deuterated samples was analyzed by Fourier self‑deconvolution followed by curve fitting using a new computer‑generated parameter procedure. The analysis accurately estimates α‑helix and β‑sheet content with an 8.6 % standard deviation relative to X‑ray structures, simultaneously resolves secondary‑structure orientation and phospholipid acyl‑chain orientation for membrane proteins, and determines the lipid/protein ratio quickly from the infrared spectrum.

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 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.