title: The kinetics of thermal stress induced denaturation of Aquaporin 0.
abstract: Aquaporin 0 (AQP0) is an integral membrane protein that facilitates water transport and cellular adhesion in the lens. Its dysfunction has been associated with cataractogenesis. Our earlier studies showed AQP0 undergoes aggregation when subjected to thermal stress and this aggregation seems to have been facilitated by mechanical agitation brought about by gentle stirring. The purpose of this study is to determine the secondary structural changes that precede aggregation and the role that α-crystallin plays in inhibiting those structural changes. Detergent solubilized calf lens AQP0 was subjected to thermal stress at 50°C for varying times. Secondary structural changes were measured by Circular Dichroism (CD) spectropolarimetry. Convex constraint analysis was used to deconvolute the CD spectra into pure component curves representing the secondary structural elements. Our results showed that under thermal stress, the α-helix content of AQP0 decreased from 50% to 7% with a concomitant increase from 0% to 52% in β-sheet content. The time-dependent loss of α-helical structure and gain of β-sheet structure appear to follow first-order kinetics with very similar values (∼30min) suggesting a single transition. In the presence of α-crystallin, this conversion to β-sheet is minimized, suggesting that the protein structure that binds to the molecular chaperone is mostly the α-helical structure of AQP0.
authors.affiliation: Department of Chemistry, University of West Georgia, Carrollton, GA 30118, USA. Electronic address: jhansen@westga.edu.
authors.forename: John E
authors.initials: JE
authors.lastname: Hansen
authors.affiliation: Department of Chemistry, University of West Georgia, Carrollton, GA 30118, USA.
authors.forename: Logan
authors.initials: L
authors.lastname: Leslie
authors.affiliation: Department of Biology, University of West Georgia, Carrollton, GA 30118, USA.
authors.forename: Satyanarayana
authors.initials: S
authors.lastname: Swamy-Mruthinti
mesh_terms: D000818:Animals; D020346:Aquaporins / Q000737:chemistry*; D002417:Cattle; D002942:Circular Dichroism; D005136:Eye Proteins / Q000737:chemistry*; D006358:Hot Temperature; D007700:Kinetics; D007908:Lens, Crystalline / Q000737:chemistry; D011489:Protein Denaturation; D017433:Protein Structure, Secondary
chemical_list: D020346:Aquaporins; D005136:Eye Proteins; C030328:aquaporin 0
keywords: Aggregation; Aquaporin 0; Circular Dichroism; Denaturation; Secondary structure; α-Crystallin