title: Conformational prerequisites for formation of amyloid fibrils from histones.
abstract: We demonstrate that bovine core histones are natively unfolded proteins in solutions with low ionic strength due to their high net positive charge at pH 7.5. Using a variety of biophysical techniques we characterized their conformation as a function of pH and ionic strength, as well as correlating the conformation with aggregation and amyloid fibril formation. Tertiary structure was absent under all conditions except at pH 7.5 and high ionic strength. The addition of trifluoroethanol or high ionic strength induced significant alpha-helical secondary structure at pH 7.5. At low pH and high salt concentration, small-angle X-ray scattering and SEC HPLC indicate the histones are present as a hexadecamer of globular subunits. The secondary structure at low pH was independent of the ionic strength or presence of TFE, as judged by FTIR. The data indicate that histones are able to adopt five different relatively stable conformations; this conformational variability probably reflects, in part, their intrinsically disordered structure. Under most of the conditions studied the histones formed amyloid fibrils with typical morphology as seen by electron microscopy. In contrast to most aggregation/amyloidogenic systems, the kinetics of fibrillation showed an inverse dependence on histone concentration; we attribute this to partitioning to a faster pathway leading to non-fibrillar self-associated aggregates at higher protein concentrations. The rate of fibril formation was maximal at low pH, and decreased to zero by pH 10. The kinetics of fibrillation were very dependent on the ionic strength, increasing with increasing salt concentration, and showing marked dependence on the nature of the ions; interestingly Gdn.HCl increased the rate of fibrillation, although much less than NaCl. Different ions also differentially affected the rate of nucleation and the rate of fibril elongation.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA.
authors.forename: Larissa A
authors.initials: LA
authors.lastname: Munishkina
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Fink
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Uversky
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006657:Histones / Q000737:chemistry* / Q000648:ultrastructure; D008854:Microscopy, Electron; D009994:Osmolar Concentration; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D017550:Spectroscopy, Fourier Transform Infrared; D014508:Urea / Q000737:chemistry
chemical_list: D000682:Amyloid; D006657:Histones; D014508:Urea