Natively unfolded state for engineering nanoscale fibrillar arrays.

A generic rationale for the fabrication of high aspect ratio fibrillar nanoscale arrays is described. The design emulates an intermittence effect observed for β-structured α-synunclein fibrils, reported herein, in a structurally unrelated α-helical fiber. The generated nanoarrays are composed of periodic nanosized segments separated at uniform distances of unfolded regions.

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes.

Single molecule fluorescent microscopy is a method for the analysis of the dynamics of biological macromolecules by detecting the fluorescence signal produced by fluorophores associated with the macromolecule. Two fluorophores located in a close proximity may result in Förster resonance energy transfer (FRET), which can be detected at the single molecule level and the efficiency of energy transfer calculated. In most cases, the experimentally observed distribution of FRET efficiency exhibits a significant width corresponding to 0.

Sequestering of p53 into DNA-protein filaments revealed by electron microscopy.

Using electron microscopy, we analyzed the interaction of bacterially expressed full-length p53, p53(1-393), and its C-terminal fragment, p53(320-393), with long (approximately 3000 bp) dsDNA in linear and supercoiled (|DeltaLk| approximately 4-6) forms containing or lacking the p53 recognition sequence (p53CON). The main structural feature of the complexes formed by either protein was a DNA-protein filament, in which two DNA duplexes are linked (synapsed) via bound protein tetramers. The efficiency of the synapse, reflected in its length and the fraction of molecules exhibiting DNA-protein filaments, was significantly modulated by the molecular form of the protein and the topological state of the DNA.

Inducing and modulating anisotropic DNA bends by pseudocomplementary peptide nucleic acids.

DNA bending is significant for various DNA functions in the cell. Here, we demonstrate that pseudocomplementary peptide nucleic acids (pcPNAs) represent a class of versatile, sequence-specific DNA-bending agents. The occurrence of anisotropic DNA bends induced by pcPNAs is shown by gel electrophoretic phasing analysis.

Role of tumor suppressor p53 domains in selective binding to supercoiled DNA.

We showed previously that bacterially expressed full-length human wild-type p53b(1-393) binds selectively to supercoiled (sc)DNA in sc/linear DNA competition experiments, a process we termed supercoil-selective (SCS) binding. Using p53 deletion mutants and pBluescript scDNA (lacking the p53 recognition sequence) at native superhelix density we demonstrate here that the p53 C-terminal domain (amino acids 347-382) and a p53 oligomeric state are important for SCS binding. Monomeric p53(361-393) protein (lacking the p53 tetramerization domain, amino acids 325-356) did not exhibit SCS binding while both dimeric mutant p53(319- 393)L344A and fusion protein GCN4-p53(347-393) were effective in SCS binding.

Nucleosome-like complex of the histone from the hyperthermophile Methanopyrus kandleri (MkaH) with linear DNA.

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.

Amyloid fibrils from the mammalian protein prothymosin alpha.

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.

Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri.

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.