title: Stimulation of insulin fibrillation by urea-induced intermediates.
abstract: Fibrillar deposits of insulin cause serious problems in implantable insulin pumps, commercial production of insulin, and for some diabetics. We performed a systematic investigation of the effect of urea-induced structural perturbations on the mechanism of fibrillation of insulin. The addition of as little as 0.5 m urea to zinc-bound hexameric insulin led to dissociation into dimers. Moderate concentrations of urea led to accumulation of a partially unfolded dimer state, which dissociates into an expanded, partially folded monomeric state. Very high concentrations of urea resulted in an unfolded monomer with some residual structure. The addition of even very low concentrations of urea resulted in increased fibrillation. Accelerated fibrillation correlated with population of the partially folded intermediates, which existed at up to 8 m urea, accounting for the formation of substantial amounts of fibrils under such conditions. Under monomeric conditions the addition of low concentrations of urea slowed down the rate of fibrillation, e.g. 5-fold at 0.75 m urea. The decreased fibrillation of the monomer was due to an induced non-native conformation with significantly increased alpha-helical content compared with the native conformation. The data indicate a close-knit relationship between insulin conformation and propensity to fibrillate. The correlation between fibrillation and the partially unfolded monomer indicates that the latter is a critical amyloidogenic intermediate in insulin fibrillation.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
authors.forename: Atta
authors.initials: A
authors.lastname: Ahmad
authors.forename: Ian S
authors.initials: IS
authors.lastname: Millett
authors.forename: Sebastian
authors.initials: S
authors.lastname: Doniach
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Uversky
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Fink
mesh_terms: D002845:Chromatography; D002942:Circular Dichroism; D019281:Dimerization; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D008854:Microscopy, Electron; D008956:Models, Chemical; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013053:Spectrophotometry; D014466:Ultraviolet Rays; D014508:Urea / Q000737:chemistry / Q000494:pharmacology*; D014965:X-Rays; D015032:Zinc / Q000737:chemistry
chemical_list: D007328:Insulin; D014508:Urea; D015032:Zinc