title: Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces.
abstract: In hydrophobic interaction chromatography (HIC), interactions between buried hydrophobic residues and HIC surfaces can cause conformational changes that interfere with separations and cause yield losses. This paper extends our previous investigations of protein unfolding in HIC chromatography by identifying protein structures on HIC surfaces under denaturing conditions and relating them to solution behavior. The thermal unfolding of three model multidomain proteins on three HIC surfaces of differing hydrophobicities was investigated with hydrogen exchange mass spectrometry (HXMS). The data were analyzed to obtain unfolding rates and Gibbs free energies for unfolding of adsorbed proteins. The melting temperatures of the proteins were lowered, but by different amounts, on the different surfaces. In addition, the structures of the proteins on the chromatographic surfaces were similar to the partially unfolded structures produced in the absence of a surface by temperature as well as by chemical denaturants. Finally, it was found that patterns of residue exposure to solvent on different surfaces at different temperatures can be largely superimposed. These findings suggest that protein unfolding on various HIC surfaces might be quantitatively related to protein unfolding in solution and that details of surface unfolding behavior might be generalized.
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904 4741, USA. Electronic address: adriangospodarek@gmail.com.
authors.forename: Adrian M
authors.initials: AM
authors.lastname: Gospodarek
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904 4741, USA.
authors.forename: Weitong
authors.initials: W
authors.lastname: Sun
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904 4741, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: O'Connell
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904 4741, USA.
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Fernandez
mesh_terms: D000327:Adsorption; D000818:Animals; D002417:Cattle; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry*; D012997:Solvents; D013499:Surface Properties
chemical_list: D011506:Proteins; D012997:Solvents
keywords: Hydrogen exchange mass spectrometry; Hydrophobic interaction chromatography; Protein unfolding; Surface induced denaturation