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Protein Science (2005), 14:2781-2792. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Undistorted structural analysis of soluble proteins by attenuated total reflectance infrared spectroscopy

Michel E. Goldberg and Alain F. Chaffotte

Unité de Repliement et Modélisation des Protéines, Department of Structural Biology and Chemistry, Institut Pasteur, Paris, France

(RECEIVED June 30, 2005; FINAL REVISION August 22, 2005; ACCEPTED August 22, 2005)

Water from the solvent very strongly absorbs light in the frequency range of interest for studying protein structure by infrared (IR) spectroscopy. This renders handling of the observation cells painstaking and time consuming, and limits the reproducibility of the measurements when IR spectroscopy is applied to proteins in aqueous solutions. These difficulties are circumvented by the use of an Attenuated Total Reflectance (ATR) accessory. However, when protein solutions are studied, ATR spectroscopy suffers from several drawbacks, the most severe being nonproportionality of the observed absorbance with the protein concentration and spectral distortions that vary from protein to protein and from sample to sample. In this study, we show (1) that the nonproportionality is due to adsorption of the protein on the ATR crystal surface; (2) that the contribution of the crystal-adsorbed protein can easily be taken into account, rendering the corrected absorbance proportional to the protein concentration; (3) that the observed variable base line distortions, likely due to changes in the penetration depth of the light beam in solutions with the refractive index that depends on the protein concentration, can be easily eliminated; and (4) that ATR IR spectra thus corrected for protein adsorption and light penetration can be used to properly analyze the secondary structure of proteins in solution.

Keywords: ATR; FTIR; proteins; secondary structure; adsorption

Abbreviations: ATR, attenuated total reflectance • FKPA, a peptidylprolyl cis–trans isomerase from Escherichia coli • FTIR, Fourier transform infrared • MBP, the maltose binding protein from Escherichia coli • PDB, the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank • TRX, the oxidized thioredoxin from Escherichia coli

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051678205.

Reprint requests to: Michel E. Goldberg, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France; e-mail: goldberg{at}pasteur.fr; fax: +33-1-40-61-30-43.


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