title: Chiral bifurcation in aggregating insulin: an induced circular dichroism study.
abstract: The structural unambiguity of folding is lost when disordered protein molecules convert into beta-sheet-rich fibrils. The resulting polymorphism of protein aggregates has been studied in the context of its biomedical consequences. Events underlying the conformational variance of amyloid fibrils, as well as physicochemical boundaries between folding and misfolding pathways, remain obscure. Bifurcation and chiral mesoscopic-scale organization of amyloid fibrils are new aspects of protein misfolding. Here we characterize bifurcation events accompanying insulin fibrillation upon intensive vortexing. Upon agitation, two types of insulin fibrils with opposite chiral senses are formed; however, predominance of either species is only stochastically determined. The uncertainty of fibrils' chiral sense holds only for fibrils grown within the physiological temperature range, while above 50 degrees C, the bifurcation is no longer observed--fibrils' chiral moieties become uniformly biased towards ligand probes, as revealed by the extrinsic Cotton effect of thioflavin T, Congo red, and molecular iodine. According to transmission electron microscopy and scanning electron microscopy data, chiral variants of insulin fibrils consist of fibrous superstructures, distinct from spherulites, formed by the protein in nonagitated solutions. Gradual dissociation of the fibrils in the presence of dimethyl sulfoxide is noncooperative and can be resolved into three distinct phases: decay of the higher-order chiral structures, breakdown of fibrils, and unfolding of intermolecular beta-sheet. The chiral aggregates are also destabilized by elution of NaCl implying that Debye screening of charged beta-sheets provided by chloride counterions is needed for sustaining their kinetic stability. At elevated temperatures, cross-seeding of agitated insulin samples with preformed fibrils revealed a chiral conflict that prevented the passing of structural features of mother seeds to daughter fibrils in a manner typical of amyloid "strains."
authors.affiliation: Institute of High Pressure Physics, Polish Academy of Sciences, Sokolowska 29/37, 01-142 Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Loksztejn
authors.forename: Wojciech
authors.initials: W
authors.lastname: Dzwolak
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D002942:Circular Dichroism; D003224:Congo Red / Q000737:chemistry; D007328:Insulin / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013696:Temperature; D013844:Thiazoles / Q000737:chemistry
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D007328:Insulin; D013844:Thiazoles; C009462:thioflavin T; D003224:Congo Red