No abstract

NF-M13 [H-(Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly)-OH], NF-M17 [H-(Glu-Glu-Lys-Gly-Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly) -OH], and their phosphorylated derivatives, representing the C-terminal phosphorylation domain of the neurofilament protein midsize subunit, have four possible binding sites for metal ions: the COO- group of glutamate, the OH group of the serine residue, the PO3H- group of phosphoserine (when present), and the COO- at the terminus of the peptide chain. The CD titration of the phosphorylated neurofilament fragments with Al3+ and Ca2+ yielded a significant conformational change that resulted in conformations containing high beta-pleated-sheet contents, which precipitate on standing (intermolecular complex). Al3+ binding to the unphosphorylated NF-M13 and NF-M17 did not exhibit this behavior. Several alanine analogues of the parent NF-M17 peptide were synthesized in order to determine the relationship between metal ions and possible binding sites. CD titration of analogues with Ca2+ indicated that the critical residues of NF-M17 for Ca(2+)-induced conformational changes, from random to beta-pleated sheet, are the N-terminal serine or both phosphorylated serines. Al(3+)-induced conformational changes suggest that the critical sites of NF-M17 yielding the beta-pleated-sheet structure are the four glutamates or phosphorylated serines, especially the C-terminal SerP. On the basis of the titration data, it is very likely that analogues with a serine in position 11 form a stable intramolecular complex with Al3+ that, however, does not result in the adoption of the beta-conformation. Back-titration with citric acid fails to reverse the Al(3+)-induced conformational changes of the phosphorylated peptides. The above results, especially the possible formation of intramolecular and intermolecular Al3+ complexes, may have relevance to the molecular mechanism, through which the neurotoxin Al3+ gives rise to the formation of neurofilament tangles.

Low molecular weight histone complexes of H2A (congruent to dimer), H2B (congruent to tetramer), H3--H4 (congruent to tetramer), H2A--H2B (congruent to dimer), and H2B--H4 (congruent to dimer) have been prepared in 2 M NaCl and neutral pH at 4 degrees C. These materials are free of nonspecific aggregate and are suitable for study by high resolution proton magnetic resonance spectroscopy. Such spectra have been recorded in aqueous solutions under conditions allowing a study of the exchangeable proton resonances of histone complexes for the first time and indicate that the structured regions are rich in hydrophobic amino acids, as well as arginine and some acidic amino acids. Most of the lysine and probably alanine residues remain in a motile, random coil-like state after formation of the complexes. It is suggested that arginine residues may be important in inter- and/or intra-subunit interactions in histone complexes.

No abstract

Nucleoprotein complexes reconstituted from calf thymus DNA and lysine-rich histone (f-1 fraction) in a variety of neutral salts and in dioxane were examined by circular dichroism. The alteration of the DNA ellipticity bands is interpreted as a reflection of conformational change of the DNA in the complexes. Gradient dialysis of DNA and f-1 in various ratios into 0.14 or 0.3 M uni-univalent salt solutions results in circular dichroic spectra which can be approximately ordered according to the lyotropic series. This sequence was found to be, in order of the effect on the DNA circular dichroic spectrum (that is, beginning the sequence with salts that alter the circular dichroism spectra the most): guanidine'HC1 > NHdC1, NH40Ac >> CsCl > KC1, KF > NaOAc, NaBr, NaF, NaCl > LiCl >> NaC104, KCNS, NaI. Most of the effect is exerted through cations.Blkali metal ions enhance the ellipticity changes of complexes to various extents. Ammonium and guanidinium ions cause the largest changes in the circular dichroism; these specific ion effects are remarkable because of the relatively low salt concentration at which they operate. Most anions (OAc-, F-, C1-, Br-) have little effect, except for C104-, CNS-, and I-, which dissociate the complexes. Very low MgClz concentration ( 5 0.006 M ) augments conformational changes; at higher concentration MgClz causes dissociation. Addition of dioxane (up to 40% by volume) enhances distortion of the DNA ellipticity. Different types of complexes result (1) from direct mixing of f-1 histone and DNA in 0.14 iM KaF, (2) from dialysis into 0.01 M NaF, and (3) from dialysis into 0.14 M NaF. Filtration experiments indicate that histone binds to DNA in a partially cooperative manner. f-1-DNA interaction is modified by salt concentration, specific salt effects, dioxane, and manner of complex formation. This complicated behavior indicates that charge attraction between histone and DNA, even when supplemented by hydrophobic bonding, is insufficient to account for the observed circular dichroic changes. Specific association of complexes may be involved, mediated by the presence of various salts.