Circuit Topology of Proteins and Nucleic Acids

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Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5’-ATCGAT-3’ sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.     

A Multi-sample Microdialysis Apparatus for Proteins and Nucleic Acids

Abstract

A multiposition microdialysis system suitable for simultaneous microsample applications (between 10 μL and 500 μL), has been developed. Each sample, contained in a specially designed microfuge dialysis tube (mDT), is dialysed independently from the other samples. Each mDT has its own membrane, and this feature allows the use of different membranes and dialysis times for different samples.

The microdialysis apparatus is kept at constant temperature by an external thermostat, avoiding the use of a cold box. The dialysis release time for small ions, a parameter used for quantitation of microdialysis efficiency, decreases from 22.9 min (for a 200 μL sample) to 7 min (for a 50 μL sample). The sample is efficiently recovered by centrifugation. Quantitative recoveries (90%) of different proteins and DNA were achieved after microdialysis by mDT.     

Covalent Interaction of Proteins and Nucleic Acids. Synthetic and Natural Nucleotide-Peptides

Abstract

The review reports data on the occurrence, structure and functions of covalent nucleotide and NA-protein complexes. Methods of synthesis of model nucleotide-peptides with phosphoester and phosphoamide bonds and their hydrolytic properties are discussed. Separate sections are devoted to methods of specific cleavage of phosphoamide and phosphoester bonds in nucleotide-peptides and their application in biochemistry.     

Emerging Methods and Applications to Decrypt Allostery in Proteins and Nucleic Acids

Many large protein-nucleic acid complexes exhibit allosteric regulation. In these systems, the propagation of the allosteric signaling is strongly coupled to conformational dynamics and catalytic function, challenging state-of-the-art analytical methods. Here, we review established and innovative approaches used to elucidate allosteric mechanisms in these complexes. Specifically, we report network models derived from graph theory and centrality analyses in combination with molecular dynamics (MD) simulations, introducing novel schemes that implement the synergistic use of graph theory with enhanced simulations methods and ab-initio MD. Accelerated MD simulations are used to construct “enhanced network models”, describing the allosteric response over long timescales and capturing the relation between allostery and conformational changes. “Ab-initio network models” combine graph theory with ab-initio MD and quantum mechanics/molecular mechanics (QM/MM) simulations to describe the allosteric regulation of catalysis by following the step-by-step dynamics of biochemical reactions. This approach characterizes how the allosteric regulation changes from reactants to products and how it affects the transition state, revealing a tense-to-relaxed allosteric regulation along the chemical step. Allosteric models and applications are showcased for three paradigmatic examples of allostery in protein-nucleic acid complexes: (i) the nucleosome core particle, (ii) the CRISPR-Cas9 genome editing system and (iii) the spliceosome. These methods and applications create innovative protocols to determine allosteric mechanisms in protein-nucleic acid complexes that show tremendous promise for medicine and bioengineering.     

Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with K_D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.     

Interactions of NBU1 IntN1 and Orf2x Proteins with Attachment Site DNA

NBU1 is a mobilizable transposon found in Bacteroides spp. Mobilizable transposons require gene products from coresident conjugative transposons for excision and transfer to recipient cells. The integration of NBU1 requires IntN1, which has been identified as a tyrosine recombinase, as well as Bacteroides host factor BHFa. Excision of NBU1 is a more complicated process, involving five element-encoded proteins (IntN1, Orf2, Orf2x, Orf3, and PrmN1) as well as a Bacteroides host factor and a cis-acting DNA sequence. Little has been known about what role the proteins play in excision, although IntN1 and Orf2x have been shown to be the only proteins absolutely required for detectable excision. To determine where IntN1 and Orf2x bind during the excision of NBU1, both proteins were partially purified and tested in DNase I footprinting experiments with the excisive attachment sites attL and attR. The results demonstrate that IntN1 binds to four core-type sites that flank the region of cleavage and strand exchange, as well as six arm-type sites. A unique feature of the system is the location of DR2a and DR2b arm-type sites immediately downstream of the attL core. The DR1a, DR1b, DR3a, and DR3b arm-type sites were shown to be required for in vitro integration of NBU1. In addition, we have identified one Orf2x binding site (O1) on attL as well as a dA+dT-rich upstream element that is required for Orf2x interactions with O1.     

Chromatin Immunoprecipitation Indirect Peaks Highlight Long-Range Interactions of Insulator Proteins and Pol II Pausing

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Changes of Nucleic Acids and Proteins in the Oocysts of Eimeria tenella during Sporulation

SYNOPSIS. The total content of DNA in Eimeria tenella , estimated at 5.8 × 10⁻¹² gm/oocyst, varies little during sporulation. Its buoyant density is 1.682 gm/cm³, reflecting a G + C content of ∼41%. Thymidine is not incorporated into any TCA insoluble fraction of sporulating oocysts, but radioactivity from [³H]uridine and [³H]deoxyuridine are incorporated into RNA at a linear rate during the first 5 hr of sporulation. The labeled RNA, found mainly in the paranuclear bodies of newly formed sporozoites, contains ∼0.15 nmole [³H]uridine/10⁶ oocysts at the completion of sporulation. One nmole of leucine is incorporated into the hot TCA insoluble fraction of 10⁶ oocysts during the first 7 hr of sporulation after an initial lag. The incorporated amino acid is mainly in the cytoplasm of the sporozoites, and an analysis by SDS-gel electrophoresis reveals most of the radioactivity in a narrow band with a molecular weight of ∼50,000 daltons. Incorporation of uridine and leucine, however, can be totally suppressed by respiratory inhibition. Further analysis of the proteins in the oocysts reveals that the total protein content remains relatively unchanged at 2.64 × 10⁻¹⁶ gm/oocyst during sporulation, but there is a shift of 13–14% of total protein from the soluble cytoplasm to the 15,000 g pellets. By polyacrylamide gel electrophoresis, a major protein band. possibly a glycoprotein, is shown in the soluble cytoplasm of unsporulated oocysts. This band disappears during sporulation.     

Methylation of Nucleic Acids and Proteins in the Liver of a Rat Treated with 3H-Methylazoxymethanol Acetate

Titration curves of ovalbumin and s-ovalbumin were compared in studies of changes in properties of ovalbumin on conversion to s-ovalbumin. Although there are 49 carboxyl groups in ovalbumin, two were not titrated in 0.25 M KCl but on conversion to s-ovalbumin. A similar change was also noted in the two carboxyl groups of ovalbumin in denaturation with guanidine hydrochloride. Liberation of carboxyl groups was noted in ovalbumin samples which were assumed to contain various amounts of intermediate and the isoelectric focusing patterns of these samples also changed. No difference was noted in the both amounts of ionizable amino groups and phenolic hydroxyl groups between ovalbumin and s-ovalbumin. This seems to show that these two groups are not concerned with the liberation of carboxyl groups during ovalbumin-s-ovalbumin transformation.     

Investigating the Comparative Biology of the Heterokonts with Nucleic Acids1

During the last decade investigations of heterokont protists utilizing molecular methods have challenged established biosystematic concepts. Most investigations emphasized the chloroplast genome or sequences from nuclear-encoded, ribosomal genes. Refinement of DNA isolation protocols, advent of “universal primers” and the polymerase chain reaction, automated sequencing and increased accessibility of DNA sequence databases have expanded data-gathering efficiency and increased dataset sizes. Because independent datasets have been easier to obtain, the testing of specific phylogenetic hypotheses has been facilitated, altering relationship concepts, primarily at phylum/class levels, and perceptions of cellular evolution. New approaches have emphasized ecological studies and extended studies to genus/species levels and poorly investigated genomes. This paper reviews studies documenting these impacts and identifies some current limitations. Additionally, new DNA sequence data from our laboratory on nuclear-encoded rDNA internal transcribed spacers and the chloroplast-encoded psb A gene suggest that these regions will provide taxonomic resolution for the Synu-rophyceae, at the class/order level and subspecies level, respectively.     

Interactions of 5-(1-Pyrenyl)-10,15,20-tris(4-methylpyridinium)porphine with Double-Helix and Triple-Helix Nucleic Acids

Abstract

5-(1 -Pyrenyl)-10,15,20-tris(4-methylpyridinium)porphine (H₂PTMPP) having a porphyrin ring and a pyrenyl substituent was synthesized. The compound H₂PTMPP bound to poly(dA)?poly(dT) double helix and poly(dA)?2poly(dT) triple helix in different styles. The results of H₂PTMPP binding to oligonucleotides, dA₁₄?dT₁₄ and dA₁₄?2dT₁₄, was also shown.

     

Role of Aromatic Amino-acid Residues in the Binding of Enzymes and Proteins to Nucleic Acids

MANY studies have been made of the specificity of interaction between nucleic acids and polypeptides, proteins and enzymes^1,2. Electrostatic forces between basic amino-acids and phosphate groups contribute to the stability of the complexes, but selective recognition requires more specific interactions which are not yet understood. The recognition of a specific region of a nucleic acid could be explained if this region has some particular conformation or if there are specific interactions between a few amino-acid residues and the bases of this region. We wish to report results which show that the aromatic amino-acids tryptophan and tyrosine can interact with nucleic acid bases in double stranded nucleic acids. They suggest that aromatic amino-acid residues of enzymes and proteins could participate in the binding to nucleic acids by intercalating between the bases and thus constraining the nucleic acid molecule to adopt a definite position with respect to the protein molecule.     

Cardiolipin Interactions with Proteins

Cardiolipins (CL) represent unique phospholipids of bacteria and eukaryotic mitochondria with four acyl chains and two phosphate groups that have been implicated in numerous functions from energy metabolism to apoptosis. Many proteins are known to interact with CL, and several cocrystal structures of protein-CL complexes exist. In this work, we describe the collection of the first systematic and, to the best of our knowledge, the comprehensive gold standard data set of all known CL-binding proteins. There are 62 proteins in this data set, 21 of which have nonredundant crystal structures with bound CL molecules available. Using binding patch analysis of amino acid frequencies, secondary structures and loop supersecondary structures considering phosphate and acyl chain binding regions together and separately, we gained a detailed understanding of the general structural and dynamic features involved in CL binding to proteins. Exhaustive docking of CL to all known structures of proteins experimentally shown to interact with CL demonstrated the validity of the docking approach, and provides a rich source of information for experimentalists who may wish to validate predictions.     

Chromatin Proteins Share Antigenic Determinants with Neurofilaments

Antigenic determinants common to distinct proteins may be unambiguously identified by the use of monoclonal antibodies. Some monoclonal antibodies to mammalian neurofilaments have recently been shown to cross-react with the neurofibrillary tangles found at high density in the brains of senile dements with Alzheimers disease (SDAT). Here, we show that these antibodies also cross-react with chromatin proteins, including the linker histones H1 and H1(0). Elevated levels of histone H1(0) have also been reported in SDAT brains.     

Biological Microchips with Hydrogel-Immobilized Nucleic Acids,Proteins, and Other Compounds: Properties and Applications in Genomics

The MAGIChip (MicroArrays of Gel-Immobilized Compounds on a chip) consists of an array of hydrophilic gel pads fixed on a hydrophobic glass surface. These pads of several picoliters to several nanoliters in volume contain gel-immobilized nucleic acids, proteins, and other compounds, as well as live cells. They are used to conduct chemical and enzymatic reactions with the immobilized compounds or samples bound to them. In the latter case, nucleic acid fragments can be hybridized, modified, and fractionated within the gel pads. The main procedures required to analyze nucleic acid sequences (PCR, detachment of primers and PCR-amplified products from a substrate, hybridization, ligation, and others) can be also performed within the microchip pads. A flexible, multipurpose, and inexpensive system has been developed to register the processes on a microchip. The system provides unique possibilities for research and biomedical applications, allowing one to register both equilibrium states and the course of reaction in real time. The system is applied to analyze both kinetic and thermodynamic characteristics of molecular interaction in the duplexes formed between nucleic acids and the probes immobilized within the microchip gel pads. Owing to the effect of stacking interaction of nucleic acids, the use of short oligonucleotides extends the possibilities of microchips for analysis of nucleic acid sequences, allowing one to employ the MALDI-TOF mass spectrometry to analyze the hybridization data. The specialized MAGIChips has been successfully applied to reveal single-nucleotide polymorphism of many biologically significant genes, to identify bacteria and viruses, to detect toxins and characterize the genes of pathogenic bacteria responsible for drug resistance, and to study translocations in the human genome. On the basis of the MAGIChip, protein microchips have been created, containing immobilized antibodies, antigens, enzymes, and many other substances, as well as microchips with gel-immobilized live cells.