RESUMO
The MkaH protein from the archaeon Methanopyrus kandleri, an unusual assembly of two histone-fold domains in a single polypeptide chain, demonstrates high structural similarity to eukaryal histones. We studied the DNA binding and self-association properties of MkaH by means of the electrophoretic mobility shift assay (EMSA), electron microscopy (EM), chemical cross-linking, and analytical gel filtration. EMSA showed an increased mobility of linear DNA complexed with MkaH protein with a maximum at a protein-DNA weight ratio (R(w)) of approximately 3; the mobility decreased at higher protein concentration. EM of the complexes formed at Rw
Assuntos
Histonas/química , Methanobacteriales/química , Nucleossomos/química , Nucleossomos/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Proteínas Arqueais/ultraestrutura , Sequência de Bases , Cristalografia por Raios X , DNA Arqueal/química , DNA Arqueal/metabolismo , DNA Arqueal/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Dimerização , Histonas/metabolismo , Histonas/ultraestrutura , Cinética , Peso Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestrutura , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/ultraestrutura , Nucleossomos/ultraestrutura , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
Mammalian prothymosin alpha, a small (12 kDa) and extremely acidic protein (pI 3.5), is a member of the growing family of 'natively' unfolded proteins. We demonstrate that at low pH ( approximately 3) and high concentrations, prothymosin alpha is capable of forming regular elongated fibrils with flat ribbon structure 4-5 nm in height and 12-13 nm in width as judged from scanning force and electron microscopy. These aggregates induced a characteristic spectral shift of thioflavin T fluorescence and their circular dichroism spectra were indicative of significant beta-sheet content, suggesting formation of classical amyloid. Our findings indicate that natively unfolded proteins may have a general propensity to form amyloid fibrils under conditions inducing partially folded conformations.
Assuntos
Peptídeos beta-Amiloides/química , Precursores de Proteínas/química , Tiazóis/análise , Timosina/análogos & derivados , Timosina/química , Benzotiazóis , Dicroísmo Circular , Corantes Fluorescentes/análise , Humanos , Concentração de Íons de Hidrogênio , Cinética , Microscopia de Força Atômica , Microscopia Eletrônica , Dobramento de Proteína , Precursores de Proteínas/isolamento & purificação , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Timosina/isolamento & purificaçãoRESUMO
Three novel DNA-binding proteins with apparent molecular masses of 7, 10 and 30 kDa have been isolated from the hyperthermophilic methanogen Methanopyrus kandleri. The proteins were identified using a blot overlay assay that was modified to emulate the high ionic strength intracellular environment of M.kandleri proteins. A 7 kDa protein, named 7kMk, was cloned and expressed in Escherichia coli. As indicated by CD spectroscopy and computer-assisted structure prediction methods, 7kMk is a substantially alpha-helical protein possibly containing a short N-terminal beta-strand. According to analytical gel filtration chromatography and chemical crosslinking, 7kMk exists as a stable dimer, susceptible to further oligomerization. Electron microscopy showed that 7kMk bends DNA and also leads to the formation of loop-like structures of approximately 43.5 +/- 3.5 nm (136 +/- 11 bp for B-form DNA) circumference. A topoisomerase relaxation assay demonstrated that looped DNA is negatively supercoiled under physiologically relevant conditions (high salt and temperature). A BLAST search did not yield 7kMk homologs at the amino acid sequence level, but based on a multiple alignment with ribbon-helix-helix (RHH) transcriptional regulators, fold features and self-association properties of 7kMk we hypothesize that it could be related to RHH proteins.