title: Congo red populates partially unfolded states of an amyloidogenic protein to enhance aggregation and amyloid fibril formation.
abstract: Congo red (CR) has been reported to inhibit or enhance amyloid fibril formation by several proteins. To gain insight into the mechanism(s) for these apparently paradoxical effects, we studied as a model amyloidogenic protein, a dimeric immunoglobulin light chain variable domain. With a range of molar ratios of CR, i.e. r = [CR]/[protein dimer], we investigated the aggregation kinetics, conformation, hydrogen-deuterium exchange, and thermal stability of the protein. In addition, we used isothermal titration calorimetry to characterize the thermodynamics of CR binding to the protein. During incubation at 37 degrees C or during thermal scanning, with CR at r = 0.3, 1.3, and 4.8, protein aggregation was greatly accelerated compared with that measured in the absence of the dye. In contrast, with CR at r = 8.8, protein unfolding was favored over aggregation. The aggregates formed with CR at r = 0 or 0.3 were typical amyloid fibrils, but mixtures of amyloid fibrils and amorphous aggregates were formed at r = 1.3 and 4.8. CR decreased the apparent thermal unfolding temperature of the protein. Furthermore, CR perturbed the tertiary structure of the protein without significantly altering its secondary structure. Consistent with this result, CR also increased the rate of hydrogen-deuterium exchange by the protein. Isothermal titration calorimetry showed that CR binding to the protein was enthalpically driven, indicating that binding was mainly the result of electrostatic interactions. Overall, these results demonstrate that at low concentrations, CR binding to the protein favors a structurally perturbed, aggregation-competent species, resulting in acceleration of fibril formation. At high CR concentration, protein unfolding is favored over aggregation, and fibril formation is inhibited. Because low concentrations of CR can promote amyloid fibril formation, the therapeutic utility of this compound or its analogs to inhibit amyloidoses is questionable.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver 80262, USA.
authors.forename: Yong-Sung
authors.initials: YS
authors.lastname: Kim
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Randolph
authors.forename: Mark C
authors.initials: MC
authors.lastname: Manning
authors.forename: Fred J
authors.initials: FJ
authors.lastname: Stevens
authors.forename: John F
authors.initials: JF
authors.lastname: Carpenter
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D002942:Circular Dichroism; D003224:Congo Red / Q000494:pharmacology*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D008854:Microscopy, Electron; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics
chemical_list: D000682:Amyloid; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D003224:Congo Red