Functionalizing DNA Origami by Triplex-Directed Site-Specific Photo-Cross-Linking.

Here, we present a cross-linking approach to covalently functionalize and stabilize DNA origami structures in a one-pot reaction. Our strategy involves adding nucleotide sequences to adjacent staple strands, so that, upon assembly of the origami structure, the extensions form short hairpin duplexes targetable by psoralen-labeled triplex-forming oligonucleotides bearing other functional groups (pso-TFOs). Subsequent irradiation with UVA light generates psoralen adducts with one or both hairpin staples leading to site-specific attachment of the pso-TFO (and attached group) to the origami with ca. 80% efficiency. Bis-adduct formation between strands in proximal hairpins further tethers the TFO to the structure and generates "superstaples" that improve the structural integrity of the functionalized complex. We show that directing cross-linking to regions outside of the origami core dramatically reduces sensitivity of the structures to thermal denaturation and disassembly by T7 RNA polymerase. We also show that the underlying duplex regions of the origami core are digested by DNase I and thus remain accessible to read-out by DNA-binding proteins. Our strategy is scalable and cost-effective, as it works with existing DNA origami structures, does not require scaffold redesign, and can be achieved with just one psoralen-modified oligonucleotide.

The organization of RNA contacts by PTB for regulation of FAS splicing.

Post-transcriptional steps of gene expression are regulated by RNA binding proteins. Major progress has been made in characterizing RNA-protein interactions, from high resolution structures to transcriptome-wide profiling. Due to the inherent technical challenges, less attention has been paid to the way in which proteins with multiple RNA binding domains engage with target RNAs. We have investigated how the four RNA recognition motif (RRM) domains of Polypyrimidine tract binding (PTB) protein, a major splicing regulator, interact with FAS pre-mRNA under conditions in which PTB represses FAS exon 6 splicing. A combination of tethered hydroxyl radical probing, targeted inactivation of individual RRMs and single molecule analyses revealed an unequal division of labour between the four RRMs of PTB. RNA binding by RRM4 is the most important for function despite the low intrinsic binding specificity and the complete lack of effect of disrupting individual RRM4 contact points on the RNA. The ordered RRM3-4 di-domain packing provides an extended binding surface for RNA interacting at RRM4, via basic residues in the preceding linker. Our results illustrate how multiple alternative low-specificity binding configurations of RRM4 are consistent with repressor function as long as the overall ribonucleoprotein architecture provided by appropriate di-domain packing is maintained.

MBNL1 and PTB cooperate to repress splicing of Tpm1 exon 3.

Exon 3 of the rat α-tropomyosin (Tpm1) gene is repressed in smooth muscle cells, allowing inclusion of the mutually exclusive partner exon 2. Two key types of elements affect repression of exon 3 splicing: binding sites for polypyrimidine tract-binding protein (PTB) and additional negative regulatory elements consisting of clusters of UGC or CUG motifs. Here, we show that the UGC clusters are bound by muscleblind-like proteins (MBNL), which act as repressors of Tpm1 exon 3. We show that the N-terminal region of MBNL1, containing its four CCCH zinc-finger domains, is sufficient to mediate repression. The same region of MBNL1 can make a direct protein-to-protein interaction with PTB, and RNA binding by MBNL promotes this interaction, apparently by inducing a conformational change in MBNL. Moreover, single molecule analysis showed that MBNL-binding sites increase the binding of PTB to its own sites. Our data suggest that the smooth muscle splicing of Tpm1 is mediated by allosteric assembly of an RNA-protein complex minimally comprising PTB, MBNL and their cognate RNA-binding sites.

Stoichiometry of a regulatory splicing complex revealed by single-molecule analyses.

Splicing is regulated by complex interactions of numerous RNA-binding proteins. The molecular mechanisms involved remain elusive, in large part because of ignorance regarding the numbers of proteins in regulatory complexes. Polypyrimidine tract-binding protein (PTB), which regulates tissue-specific splicing, represses exon 3 of alpha-tropomyosin through distant pyrimidine-rich tracts in the flanking introns. Current models for repression involve either PTB-mediated looping or the propagation of complexes between tracts. To test these models, we used single-molecule approaches to count the number of bound PTB molecules both by counting the number of bleaching steps of GFP molecules linked to PTB within complexes and by analysing their total emissions. Both approaches showed that five or six PTB molecules assemble. Given the domain structures, this suggests that the molecules occupy primarily multiple overlapping potential sites in the polypyrimidine tracts, excluding propagation models. As an alternative to direct looping, we propose that repression involves a multistep process in which PTB binding forms small local loops, creating a platform for recruitment of other proteins that bring these loops into close proximity.

The transition in spliceosome assembly from complex E to complex A purges surplus U1 snRNPs from alternative splice sites.

Spliceosomes are assembled in stages. The first stage forms complex E, which is characterized by the presence of U1 snRNPs base-paired to the 5' splice site, components recognizing the 3' splice site and proteins thought to connect them. The splice sites are held in close proximity and the pre-mRNA is committed to splicing. Despite this, the sites for splicing appear not to be fixed until the next complex (A) forms. We have investigated the reasons why 5' splice sites are not fixed in complex E, using single molecule methods to determine the stoichiometry of U1 snRNPs bound to pre-mRNA with one or two strong 5' splice sites. In complex E most transcripts with two alternative 5' splice sites were bound by two U1 snRNPs. However, the surplus U1 snRNPs were displaced during complex A formation in an ATP-dependent process requiring an intact 3' splice site. This process leaves only one U1 snRNP per complex A, regardless of the number of potential sites. We propose a mechanism for selection of the 5' splice site. Our results show that constitutive splicing components need not be present in a fixed stoichiometry in a splicing complex.

Defining the roles and interactions of PTB.

PTB (polypyrimidine tract-binding protein) is an abundant and widely expressed RNA-binding protein with four RRM (RNA recognition motif) domains. PTB is involved in numerous post-transcriptional steps in gene expression in both the nucleus and cytoplasm, but has been best characterized as a regulatory repressor of some ASEs (alternative splicing events), and as an activator of translation driven by IRESs (internal ribosome entry segments). We have used a variety of approaches to characterize the activities of PTB and its molecular interactions with RNA substrates and protein partners. Using splice-sensitive microarrays we found that PTB acts not only as a splicing repressor but also as an activator, and that these two activities are determined by the location at which PTB binds relative to target exons. We have identified minimal splicing repressor and activator domains, and have determined high resolution structures of the second RRM domain of PTB binding to peptide motifs from the co-repressor protein Raver1. Using single-molecule techniques we have determined the stoichiometry of PTB binding to a regulated splicing substrate in whole nuclear extracts. Finally, we have used tethered hydroxyl radical probing to determine the locations on viral IRESs at which each of the four RRM domains bind. We are now combining tethered probing with single molecule analyses to gain a detailed understanding of how PTB interacts with pre-mRNA substrates to effect either repression or activation of splicing.

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.

Transcription arrest caused by long nascent RNA chains.

The transcription process is highly processive. However, specific sequence elements encoded in the nascent RNA may signal transcription pausing and/or termination. We find that under certain conditions nascent RNA chains can have a strong and apparently sequence-independent inhibitory effect on transcription. Using phage T3 RNA polymerase (T3 RNAP) and covalently closed circular (cccDNA) DNA templates that did not contain any strong termination signal, transcription was severely inhibited after a short period of time. Less than approximately 10% residual transcriptional activity remained after 10 min of incubation. The addition of RNase A almost fully restored transcription in a dose dependent manner. Throughout RNase A rescue, an elongation rate of approximately 170 nt/s was maintained and this velocity was independent of RNA transcript length, at least up to 6 kb. Instead, RNase A rescue increased the number of active elongation complexes. Thus transcription behaved as an all-or-none process. The mechanism of transcription inhibition was explored using electron microscopy and further biochemical experiments. The data suggest that multiple mechanisms may contribute to the observed effects. Part of the inhibition can be ascribed to the formation of R-loops between the nascent RNA and the DNA template, which provides "roadblocks" to trailing T3 RNAPs. Based on available literature we discuss possible in vivo implications of the results.

LPS receptor (CD14): a receptor for phagocytosis of Alzheimer's amyloid peptide.

The amyloid beta peptide 42 (Abeta(42)) plays a key role in neurotoxicity in Alzheimer's disease. Mononuclear phagocytes, i.e. microglia, have the potential to clear Abeta by phagocytosis. Recently, the lipopolysaccharide (LPS) receptor CD14 was shown to mediate phagocytosis of bacterial components and furthermore to contribute to neuroinflammation in Alzheimer's disease. Here, we investigated whether this key innate immunity receptor can interact with Abeta(42) and mediate phagocytosis of this peptide. Using flow cytometry, confocal microscopy and two-photon fluorescence lifetime imaging (FLIM) combined with fluorescence resonance energy transfer (FRET), we demonstrated a direct molecular interaction in the range of a few nanometers between Abeta(42) and CD14 in human CD14-transfected Chinese hamster ovary cells. Investigations using cells that were genetically deficient for this receptor showed that in <30 minutes exogenous Abeta(42) added to cultured primary microglial cells was phagocytosed into the cytoplasmic compartment in a CD14-dependent manner. This phagocytosis occurred at Abeta(42) concentration ranges that were considerably lower than the threshold to activate a cellular inflammatory reaction. In contrast, there was no association of CD14 to microglial internalization of microbeads. In complementary clinical experiments, we detected a pronounced CD14 immunoreactivity on parenchymal microglia spatially correlated to characteristic Alzheimer's disease lesion sites in brain sections of Alzheimer's disease patients but not in brain sections of control subjects. By showing a close interaction between CD14 and Abeta(42), demonstrating a direct role of CD14 in Abeta(42) phagocytosis, and detecting CD14-specific staining in brains of Alzheimer's disease patients, our results indicate a role of the LPS receptor in the pathophysiology of Alzheimer's disease, which could be of therapeutic relevance.

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.

Double-stranded DNA stimulates the fibrillation of alpha-synuclein in vitro and is associated with the mature fibrils: an electron microscopy study.

Filamentous aggregates formed by alpha-synuclein are a prominent and presumably key etiological factor in Parkinson's and other neurodegenerative diseases characterized by motor disorders. Numerous studies have demonstrated that various environmental and intracellular factors affect the fibrillation properties of alpha-synuclein, e.g. by accelerating the process of assembly. Histones, the major component and constituent of chromatin, interact specifically with alpha-synuclein and enhance its fibrillation significantly. Here, we report that another component of chromatin, double-stranded DNA (dsDNA), either linear or supercoiled, also interacts with wild-type alpha-synuclein, leading to a significant stimulation of alpha-synuclein assembly into mature fibrils characterized by a reduced lag phase. In general, the morphology of the fibrils remains unchanged in the presence of linear dsDNA. Electron microscopy reveals that DNA forms various types of complexes upon association with the fibrils at their surface without distortion of the double-helical structure. The existence of these complexes was confirmed by the electrophoresis, which also demonstrated that a fraction of the associated DNA was resistant to digestion by restriction endonucleases. Fibrils assembled from the alpha-synuclein mutants A30P and A53T and the C-terminally truncated variants (encoding amino acid residues 1-108 or 1-124) also form complexes with linear dsDNA. Possible mechanisms and implications of dsDNA-alpha-synuclein interactions are discussed.

Rapid self-assembly of alpha-synuclein observed by in situ atomic force microscopy.

Self-assembly of alpha-synuclein resulting in protein aggregates of diverse morphology has been implicated in the pathogenesis of Parkinson's disease and other neurodegenerative disorders known as synucleinopathies. Apart from its biomedical relevance, this aggregation process is representative of the interconversion of an unfolded protein into nanostructures with typical amyloid features. We have used in situ tapping mode atomic force microscopy to continuously monitor the self-assembly of wild-type alpha-synuclein, its disease-related mutants A30P and A53T, and the C-terminally truncated variant alpha-synuclein(1-108). Different aggregation modes were observed depending on experimental conditions, i.e. pH, protein concentration, polyamine concentration, temperature and the supporting substrate. At pH 7.5, in the absence of the biogenic polyamines spermidine or spermine, elongated sheets 1.1(+/-0.2)nm in height and presumably representing individual beta-sheet structures, were formed on mica substrates within a few minutes. Their orientation was directed by the crystalline substructure of the substrate. In contrast, sheet formation was not observed with hydrophobic highly oriented pyrolytic graphite substrates, suggesting that negatively charged surfaces promote alpha-synuclein self-assembly. In the presence of spermidine or spermine 5.9(+/-1.0)nm high spheroidal structures were preferentially formed, sharing characteristics with similar structures previously reported for several amyloidogenic proteins and linked to neurotoxicity. alpha-Synuclein spheroid formation depended critically on polyamine binding to the C terminus, revealing a promoting effect of the C terminus on alpha-synuclein assembly in the bound state. In rare cases, fibril growth from spheroids or preformed aggregates was observed. At pH 5.0, fibrils were formed initially and incorporated into amorphous aggregates in the course of the aggregation process, providing evidence for the potential of amyloid fibril surfaces to act as nucleation sites in amorphous aggregation. This study provides a direct insight into different modes of alpha-synuclein self-assembly and identifies key factors modulating the aggregation process.

Dependence of alpha-synuclein aggregate morphology on solution conditions.

Alpha-synuclein is the major component of Lewy bodies and Lewy neurites, which are granular and filamentous protein inclusions that are the defining pathological features of several neurodegenerative conditions such as Parkinson's disease. Fibrillar aggregates formed from alpha-synuclein in vitro resemble brain-derived material, but the role of such aggregates in the etiology of Parkinson's disease and their relation to the toxic molecular species remain unclear. In this study, we investigated the effects of pH and salt concentration on the in vitro assembly of human wild-type alpha-synuclein, particularly with regard to aggregation rate and aggregate morphology. Aggregates formed at pH 7.0 and pH 6.0 in the absence of NaCl and MgCl(2) were fibrillar; the pH 6.0 fibrils displayed a helical twist, as clearly evident by scanning force and electron microscopy. Incubations at pH 7.0 remained transparent during the process of aggregation and exhibited strong thioflavin-T and weak 8-anilino-1-naphthalenesulfonate (ANS) binding; furthermore, they were efficient in seeding fibrillization of fresh solutions. In contrast, incubating alpha-synuclein at low pH (pH 4.0 or pH 5.0) resulted in the rapid formation of turbid suspensions characterized by strong ANS binding, reduced thioflavin-T binding and reduced seeding efficiency. At pH 4.0, fibril formation was abrogated; instead, very large aggregates (dimensions approximately 100 microm) of amorphous appearance were visible by light microscopy. As with acidic conditions, addition of 0.2M NaCl or 10mM MgCl(2) to pH 7.0 incubations led to a shorter aggregation lag time and formation of large, amorphous aggregates. These results demonstrate that the morphology of alpha-synuclein aggregates is highly sensitive to solution conditions, implying that the fibrillar state does not necessarily represent the predominant or most functionally significant aggregated state under physiological conditions.

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.

Clustering and diversity of fluctuations for proteins.

BACKGROUND: Protein topology plays a key role in various types of interactions. Topological constraints of a protein are defined by a contact map. We studied the fluctuations of proteins with use of a new approach based on contact map. METHODS: An annealing algorithm is used to generate a 3-dimensional protein structure from the contact map. First, we study the properties of structural elements based on fluctuations by adding individual structures (domains or subdomains). Thereafter, we focus on the building block of proteins in terms of fluctuations. RESULTS: To verify our hypothesis, we analyzed the pattern of fluctuations for chymotrypsin inhibitor-2 (CI2) by unstructuring (melting) of subregions. The data show different patterns of fluctuations for the unstructured CI2 relative to that calculated for the intact protein. CONCLUSION: Our approach introduces a new concept for classifying building blocks of proteins based on thermal fluctuations.

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.

Structural characterization of copper(II) binding to alpha-synuclein: Insights into the bioinorganic chemistry of Parkinson's disease.

The aggregation of alpha-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. We demonstrate here that Cu(II) ions are effective in accelerating AS aggregation at physiologically relevant concentrations without altering the resultant fibrillar structures. By using numerous spectroscopic techniques (absorption, CD, EPR, and NMR), we have located the primary binding for Cu(II) to a specific site in the N terminus, involving His-50 as the anchoring residue and other nitrogen/oxygen donor atoms in a square planar or distorted tetragonal geometry. The carboxylate-rich C terminus, originally thought to drive copper binding, is able to coordinate a second Cu(II) equivalent, albeit with a 300-fold reduced affinity. The NMR analysis of AS-Cu(II) complexes reveals the existence of conformational restrictions in the native state of the protein. The metallobiology of Cu(II) in Parkinson's disease is discussed by a comparative analysis with other Cu(II)-binding proteins involved in neurodegenerative disorders.