title: Probing the conformation and orientation of adsorbed enzymes using side-chain modification.
abstract: The bioactivity of enzymes that are adsorbed on surfaces can be substantially influenced by the orientation of the enzyme on the surface and adsorption-induced changes in the enzyme's structure. Circular dichroism (CD) is a powerful method for observing the secondary structure of proteins; however, it provides little information regarding the tertiary structure of a protein or its adsorbed orientation. In this study, we developed methods using side-chain-specific chemical modification of solvent-exposed tryptophan residues to complement CD spectroscopy and bioactivity assays to provide greater detail regarding whether changes in enzyme bioactivity following adsorption are due to adsorbed orientation and/or adsorption-induced changes in the overall structure. These methods were then applied to investigate how adsorption influences the bioactivity of hen egg white lysozyme (HEWL) and glucose oxidase (GOx) on alkanethiol self-assembled monolayers over a range of surface chemistries. The results from these studies indicate that surface chemistry significantly influences the bioactive state of each of these enzymes but in distinctly different ways. Changes in the bioactive state of HEWL are largely governed by its adsorbed orientation, while the bioactive state of adsorbed GOx is influenced by a combination of both adsorbed orientation and adsorption-induced changes in conformation.
authors.affiliation: Department of Bioengineering, Clemson University Genomics Institute, Clemson University, Clemson, SC 29634, USA.
authors.forename: Kenan P
authors.initials: KP
authors.lastname: Fears
authors.forename: Balakrishnan
authors.initials: B
authors.lastname: Sivaraman
authors.forename: Gary L
authors.initials: GL
authors.lastname: Powell
authors.forename: Yonnie
authors.initials: Y
authors.lastname: Wu
authors.forename: Robert A
authors.initials: RA
authors.lastname: Latour
mesh_terms: D000327:Adsorption; D002942:Circular Dichroism; D013058:Mass Spectrometry; D008958:Models, Molecular; D009113:Muramidase / Q000032:analysis / Q000737:chemistry*; D011487:Protein Conformation; D013499:Surface Properties; D014364:Tryptophan / Q000032:analysis / Q000737:chemistry*
chemical_list: D014364:Tryptophan; C421864:hen egg lysozyme; D009113:Muramidase