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 0.07-0.2 (on a scale of 0-1). Here, we present a general approach describing the analysis of experimental data for a DNA/RNA duplex. We have found that for a 15 bp duplex with Cy3 and Cy5 fluorophores attached to the opposite ends of the helix, the width of the energy transfer distribution is mainly determined by the photon shot noise and the orientation factor, whereas the variation of inter-dye distances plays a minor role.

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. 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.

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. Supershifting of p53(320-393)-scDNA complexes with monoclonal antibodies revealed that the amino acid region 375-378, constituting the epitope of the Bp53-10.1 antibody, plays a role in binding of the p53(320-393) protein to scDNA. Using electron microscopy we observed p53-scDNA nucleoprotein filaments produced by all the C-terminal proteins that displayed SCS binding in the gel electrophoresis experiments; no filaments formed with the monomeric p53(361- 393) protein. We propose a model according to which two DNA duplexes are compacted into p53-scDNA filaments and discuss a role for filament formation in recombination.

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. With linear DNA, the synapse yield promoted by the C-terminus fragment was very low, but the full-length protein was effective in linking noncontiguous duplexes, leading to the formation of intramolecular loops constrained at their bases by short regions of synapsed DNA duplexes. When the linear DNA contained p53CON, regions of preferential sequence, i.e., encompassing p53CON and probably p53CON-like sequences, were predominantly synapsed, indicating a sequence specificity of the p53 core domain. With scDNA, the synapse yield was significantly higher compared to the linear counterparts and was weakly dependent on the sign of superhelicity and presence or absence of p53CON. However, the full-length protein was more effective in promoting DNA synapses compared to the C-terminal fragment. The overall structure of the DNA-protein filaments was apparently similar for either protein form, although the apparent width differed slightly (approximately 7-9 nm and approximately 10-12 nm for p53(320-393) and p53(1-393), respectively). No distortion of the DNA helices involved in the synapse was found. We conclude that the structural similarity of DNA-protein filaments observed for both proteins is attributable mainly to the C-terminus, and that the yield is dictated by the specific and possibly nonspecific interactions of the core domain in combination with DNA topology. Possible implications for the sequestering of p53 in DNA-protein filaments are discussed.

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. 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.

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. EM of the complexes formed at Rw or=9) thickened compact nucleoprotein structures were observed; no individual loops were seen within the complexes. Gel filtration chromatography and chemical fixation indicated that in the absence of DNA the dominant form of the MkaH in solution, unlike other archaeal histones, is a stable dimer (pseudo-tetramer of the histone-fold domain) apparently resembling the eukaryal (H3-H4)(2) tetramer. Similarly, dimers are the dominant form of the protein interacting with DNA. The properties of MkaH supporting the assignment of its intermediate position between other archaeal and eukaryal histones are discussed.

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 ß-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. These regions can be targeted for conformational binding and refolding with metal nanoparticle-peptide conjugates for the conversion of fibrillar arrays into nanoparticle arrays. The introduced concept opens new strategies for engineering novel nanoscale materials and devices.

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. The magnitude of DNA bending is determined by circular permutation assay and by electron microscopy, with good agreement of calculated mean values between both methods. Binding of a pair of 10-meric pcPNAs to its target DNA sequence results in moderate DNA bending with a mean value of 40-45 degrees, while binding of one self-pc 8-mer PNA to target DNA yields a somewhat larger average value of the induced DNA bend. Both bends are found to be in phase when the pcPNA target sites are separated by distances of half-integer numbers of helical turns of regular duplex DNA, resulting in an enhanced DNA bend with an average value in the range of 80-90 degrees. The occurrence of such a sharp bend within the DNA double helix is confirmed and exploited through efficient formation of 170-bp-long DNA minicircles by means of dimerization of two bent DNA fragments. The pcPNAs offer two main advantages over previously designed classes of nonnatural DNA-bending agents: they have very mild sequence limitations while targeting duplex DNA and they can easily be designed for a chosen target sequence, because their binding obeys the principle of complementarity. We conclude that pcPNAs are promising tools for inducing bends in DNA at virtually any chosen site.