Determination of the secondary structure of proteins in different environments by FTIR-ATR spectroscopy and PLS regression (original) (raw)

The secondary structures of proteins (α-helical, β-sheet, β-turn, and random coil) in the solid state and when bound to polymer beads, containing immobilized phenyl and butyl ligands such as those as commonly employed in hydrophobic interaction chromatography, have been investigated using FTIR-ATR spectroscopy and partial least squares (PLS) methods. Proteins with known structural features were used as models, including 12 proteins in the solid state and 7 proteins adsorbed onto the hydrophobic surfaces. A strong PLS correlation was achieved between predictions derived from the experimental data for 4 proteins adsorbed onto the phenyl-modified beads and reference data obtained from the X-ray crystallographic structures with r2 values of 0.9974, 0.9864, 0.9924, and 0.9743 for α-helical, β-sheet, β-turn, and random coiled structures, respectively. On the other hand, proteins adsorbed onto the butyl sorbent underwent greater secondary structural changes compared to the phenyl sorbent as evidenced from the poorer PLS r2 values (r2 are 0.9658, 0.9106, 0.9571, and 0.9340). The results thus indicate that the secondary structures for these proteins were more affected by the butyl sorbent, whereas the secondary structure remains relatively unchanged for the proteins adsorbed onto the phenyl sorbent. This study has important ramifications for understanding the nature of protein secondary structural changes following adsorption onto hydrophobic sorbent surfaces. This knowledge could also enable the development of useful protocols for enhancing the chromatographic purification of proteins in their native bioactive states. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 895–905, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com