UNCORRECTED PROOF Conformational Prerequisites for Formation of Amyloid Fibrils from Histones Larissa A. Munishkina 1 , Anthony L. Fink 1 * and Vladimir N. Uversky 1,2 * 1 Department of Chemistry and Biochemistry, University of California, Santa Cruz California 95064, USA 2 Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino Moscow Region 142292, Russia 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 a-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 vari- ability 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. q 2004 Published by Elsevier Ltd. Keywords: amyloid fibrils; histones; natively unfolded; ionic strength; conformation; secondary structure *Corresponding authors Introduction A number of human diseases originate from the deposition of stable, ordered, filamentous protein aggregates, commonly referred to as amyloid fibrils. These diseases include numerous amyl- oidoses and many neurodegenerative disorders. In each pathological state, a specific protein or protein fragment changes from its natural soluble form into insoluble fibrils, which accumulate in a variety of organs and tissues. 1–6 More than 20 different proteins have been found to be involved in protein deposition diseases. Furthermore, many proteins unrelated to disease have now been shown to form fibrils in vitro, 6 suggesting that the propensity to fibrillate is a generic property of the polypeptide chain. 7–9 There is little or no structural similarity among proteins that form amyloid fibrils: prior to fibrillation, they may be rich in b-sheet, a-helix, b-helix, or contain both a-helices and b-sheets. They may be well folded or natively unfolded (intrinsi- cally unstructured) proteins. 6,10–13 Despite these differences in the initial conformation, all the amyloid fibrils formed (both associated with protein deposition diseases and disease-unrelated), in vitro or isolated from different pathologies, display similar morphologies. Typically mature 0022-2836/$ - see front matter q 2004 Published by Elsevier Ltd. Abbreviations used: SAXS, small angle X-ray scattering; IRE, internal reflectance element; CD, circular dichroism; PAGE, polyacrylamide gel electrophoresis; ThT, Thioflavin T; MALS, multiangle light-scattering. E-mail addresses of the corresponding authors: fink@chemistry.ucsc.edu; uversky@hydrogen.ucsc.edu YJMBI 56520—6/8/2004—13:22—AMADEN—115170—XML – pp. 1–20/APPS doi:10.1016/j.jmb.2004.06.094 J. Mol. Biol. (2004) xx, 1–20 DTD 5 ARTICLE IN PRESS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126