Cross-linking of ribosomal proteins by 4-(6-formyl-3-3-azidophenoxy)butyrimidate, a heterobifunctional, cleavable cross-linker.

For the identification of neighbor relationships between proteins in biological systems 4-(6-formyl-3-azidophenoxy)butyrimidate (FAPB-imidate), a heterobifunctional, cleavable cross-linker was synthesized. The reagent has an imido ester at one end, which is used for the attachment to amino groups of a specific protein whose environment has to be characterized. At the other end, the reagent has both an azido and an aldehyde group. The azido group can be used to cross-link the protein photochemically to a variety of chemical groups of neighboring proteins. The aldehyde group is able to cross-link the protein by reductive alkylatin to amino groups of neighboring proteins. In both cases, the cross-linker can be made radioactive with NaB3H4. the cross-linked complexes can be split at the band originating from the imidate group by treatment with ammonia. Hereby, the radioactive cross-linker remains covalently attached to the unknown neighboring protein, which can be therefore easily identified. In order to explore the usefulness of FAPB-imidate as a cross-linking agent, the compound was attached to ribosomal protein L7. With this modified L7, the existence of the well-known complex between L7 and ribosomal protein L10 could be demonstrated by the photochemical procedure. By the chemical procedure, the presence of dimers of L7 in solution could be shown.     

Photochemical crosslinking of protein and DNA in chromatin. II. Synthesis and application of psoralen-cystamine-arylazido photocrosslinking reagents

The synthesis and testing of a new type of nucleic acid-protein photocrosslinking reagent is described. The reagents are composed of a psoralen ligand for nucleic acid photoattachment, which is linked to an azidobenzoyl group, for protein photoattachment. The linker contains a disulfide bridge which can be opened by reduction with mercaptans. The reagents were tested in a chromatin system, where it was found that they induced cleavable crosslinks between the histones and the DNA upon irradiation with long-wavelength ultraviolet light (lambda greater than 300 nm).     

RNA-protein neighbourhoods of the ribosome obtained by crosslinking.

It is possible to crosslink protein to nucleic acid with diepoxybutane which reacts mainly with the N-7 of guanine as well as the thiol and aminogroups in protein; the resulting crosslinked complexes are cleavable. When this reagent is applied to ribosomes, even under conditions that minimize the extent of reaction, at least half of the ribosomal proteins can be recovered crosslinked to one or other of the major ribosomal RNA species. In addition, one fourth of the proteins can be recovered crosslinked to the RNA of the heterologous ribosomal subunit. Some of the remaining proteins may also be crosslinkable to the RNA, but additional experiments are required to confirm this.     

A class of cleavable heterobifunctional reagents for thiol-directed high-efficiency protein crosslinking: synthesis and application to the analysis of protein contact sites in mammalian ribosomes

The synthesis of a new class of cleavable crosslinking reagents is described. The primary function, a ring-substituted maleimide, binds selectively and very efficiently at low pH to cysteine-containing protein sequences. At increased pH the secondary function, an N-hydroxysuccinimide ester of a ring-attached carboxyl group, becomes reactive against adjacent amino groups. The spacer, azobenzene, is readily cleaved by reduction with dithionite provided that a hydroxyl group is included in the ring system. By altering the relative positions of the reactive groups the range of crosslinking can be varied within approximately 8-12 A. After degradation of the crosslinked proteins by optional methods the contact sequences are readily identified by diagonal electrophoresis. By radiolabeling the carboxyl group of the reagent the order of binding of the proteins can be established, and the secondarily attached protein and its contact sequences can be recognized even in trace amounts. The usefulness of the reagents is illustrated by the selective, high-efficiency crosslinking of mammalian ribosomal proteins and the identification of their contact fragments as obtained by CNBr degradation.     

Syntheses of Isoquinolines by Condensation of Dopamine with Carbonyl Compounds

A cleavable photoactivable heterobifunctional reagent, the N-hydroxysuccinimide ester of 1- azido-5-naphthalene sulfonyl S-carboxymethylthiocysteamine (NHS-ANS-CTC), was synthesized and characterized. The reagent was applicable to the group-directed modification of protein ligands, such as an invertebrate lectin and a trypsin inhibitor. The modified ligands bound to rabbit erythrocyte ghosts and trypsin, respectively. Upon exposure to ultraviolet light, the modified ligands reacted with their binding proteins to form cross-linked fluorescent products. The cross-linked fluorescent complexes were readily cleaved by reducing the disulfide bond of the reagent, leaving the fluorescent probe on the binding proteins. The photolabeled binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The fluorescence intensity of the fluorescent probe was enhanced by 4~8 times to improve sensitivity when the SDS-gel was dehydrated with methanol. This phenomenon was also observed with the proteins labeled with 1-dimethylamino-5-naphthalene sulfonyl chloride.     

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.     

Selective high-efficiency cross-linking of mammalian ribosomal proteins with cleavable thiol-directed heterobifunctional reagents: identification and binding directions of major protein complexes

Rat liver and mouse ascitic tumour ribosomal proteins are cross-linked selectively in good yield with the newly developed cleavable heterobifunctional reagents 2-(4-hydroxy-2-maleimidophenylazo)benzoic acid N-hydroxysuccinimide ester (reagent A) and 4-(4-hydroxy-3-maleimidophenylazo)[carboxyl-14C]benzoic acid N-hydroxysuccinimide ester (reagent B). The primary function of the reagents, an N-aroylated maleimide, binds quantitatively at low pH to accessible cysteine groups. After eliminating the free reagent, the pH is increased to make the secondary function, a juxtanuclear aroyl ester, reactive against neighboring amino groups, essentially lysine. The spacer, 4-phenylazophenol, is readily cleaved by reduction with dithionite. The ranges of cross-linking of the two reagents are approx. 8 and 12 A, respectively. Using the radiolabelled reagent B the secondarily attached protein (and its contact sequence) is made recognizable even in trace amounts. The order of binding of the interacting proteins is thereby established. The two reagents produce similar, but not identical, patterns of selective cross-linking. The following protein complexes are readily observed after conventional staining. With reagent A: S8-S11, L4-L14, L4-L18, L6-L29 and L21-L18a. With the radioactive, longer-range reagent B: L4 ---- L13a, L4 ---- L18, L4 ---- L18a, L4 ---- L26, L6 ---- L29, L14 ---- L13a, L21 ---- L18a and L27 ---- L30 (arrows indicating the direction of binding). Ternary and quaternary complexes are also obtained, especially of the large protein L4. With both reagents a protein designated L6' is cross-linked to L23. The predominant cross-linked complexes can be obtained on a preparative scale for isolation and characterization of contact sequences by optional fragmentation and fractionation methods.     

An electron microscopic method for the mapping of proteins attached to nucleic acids.

An electron microscopic method for demonstrating the presence of and mapping the positions of proteins specifically bound to nucleic acids is described. The nucleic acid-protein complex is treated with dinitrofluorobenzene under conditions such that dinitrophenyl (DNP) groups are attached to nucleophilic groups on the protein, with only a low level of random attachment to the nuclei acid. This product is treated with rabbit anti-DNP IgG. The position of the protein-(DNP)n(IgG)m complex on the nucleic acid strand can be observed by electron microscopy by protein free spreading methods and, in many cases, by cytochrome-c spreading. If necessary for visualization by the latter method, the size of the labeled region can be increased by treatment with goat anti-rabbit IgG. High efficiency of electron microscopic labeling is achieved. Examples studied are: the adenovirus-2 DNA terminal protein, a protein covalently bound to SV40 DNA, DNA polymerase I bound to DNA, E. coli RNA polymerase bound to T7 DNA, and proteins UV crosslinked to avian sarcoma virus RNA.     

Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions

Interactions between proteins and nucleic acids are frequently analyzed using electrophoretic mobility shift assays (EMSAs). This technique separates bound protein:nucleic acid complexes from free nucleic acids by electrophoresis, most commonly using polyacrylamide gels. The current study utilizes recent advances in agarose gel electrophoresis technology to develop a new EMSA protocol that is simpler and faster than traditional polyacrylamide methods. Agarose gels are normally run at low voltages (∼10 V/cm) to minimize heating and gel artifacts. In this study we demonstrate that EMSAs performed using agarose gels can be run at high voltages (≥20 V/cm) with 0.5 × TB (Tris-borate) buffer, allowing for short run times while simultaneously yielding high band resolution. Several parameters affecting band and image quality were optimized for the procedure, including gel thickness, agarose percentage, and applied voltage. Association of the siRNA-binding protein p19 with its target RNA was investigated using the new system. The agarose gel and conventional polyacrylamide gel methods generated similar apparent binding constants in side-by-side experiments. A particular advantage of the new approach described here is that the short run times (5–10 min) reduce opportunities for dissociation of bound complexes, an important concern in non-equilibrium nucleic acid binding experiments.     

Synthesis of a cleavable protein-crosslinking reagent for the investigation of ribosome structure.

This communication describes a simple method for synthesizing cleavable bifunctional imido esters of different chain lengths. These reagents, which form covalent crosslinks between lysine residues of proteins, contain a disulfide bond which is cleaved under mild conditions by reducing agents such as 2-mercaptoethanol. The reagents are synthesized via the dithiobisnitrile which is prepared in high yield by reacting the appropriate omega-activated nitrile with sodium polysulfide and is then converted quantitatively to the diimidate. Three such reagents were prepared: dimethyl 3.3'-dithiobispropionimidate, dimethyl 4,4'-dithiobisbutyrimidate, and dimethyl 6-6'-dithiobiscaproimidate. The first was synthesized from acrylonitrile, and the others from the appropriate omega-bromonitriles. Experiments with the bispropionimidate and a test protein, pancreatic ribonuclease, have shown the reagent to be effective in producing multimeric crosslinked complexes, from which monomeric proteins can recovered after treatment with 2-mercaptoethanol. The reagents are suitable for studies of ribosomal structure.     

Specific arginine mediated RNA recognition

In many biological systems substantial roles are played by interactions between amino acids and RNA. Among amino acids L-arginine seems to be particularly relevant, because the guanidinium group of arginine side chain can potentially form five hydrogen bonds with appropriately positioned acceptor groups of RNA. Extensive studies reveal that specific arginine recognition is achieved by many different RNAs over a broad range of binding affinities. Arginine is frequently found among amino acids in the nucleic acid-binding motifs in various proteins. For example, specific binding of the HIV-1 Tat protein to its RNA site (TAR) is mediated by a single arginine residue. Free arginine can be also bound by the guanosine site in the group I Tetrahymena ribosomal RNA intron catalytic centre, as well as by numerous RNA motifs, called arginine aptamers, which have been selected in vitro.     

Reconstitution of nucleoprotein complexes with mammalian heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins

Newly transcribed heterogeneous nuclear RNA (hnRNA) in the eucaryote cell nucleus is bound by proteins, giving rise to large ribonucleoprotein (RNP) fibrils with an inherent substructure consisting largely of relatively homogeneous approximately 20-nm 30S particles, which contain core polypeptides of 34,000-38,000 mol wt. To determine whether this group of proteins was sufficient for the assembly of the native beaded nucleoprotein structure, we dissociated 30S hnRNP purified from mouse ascites cells into their component proteins and RNA by treatment with the ionic detergent sodium deoxycholate and then reconstituted this complex by addition of Triton X-100 to sequester the deoxycholate. Dissociation and reassembly were assayed by sucrose gradient centrifugation, monitoring UV absorbance, protein composition, and radiolabeled nucleic acid, and by electron microscopy. Endogenous RNA was digested and reassembly of RNP complexes carried out with equivalent amounts of exogenous RNA or single-stranded DNA. These complexes are composed exclusively of groups of n 30S subunits, as determined by sucrose gradient and electron microscope analysis, where n is the length of the added nucleic acid divided by the length of nucleic acid bound by one native 30S complex (about 1,000 nucleotides). When the nucleic acid: protein stoichiometry in the reconstitution mixture was varied, only complexes composed of 30S subunits were formed; excess protein or nucleic acid remained unbound. These results strongly suggest that core proteins determine the basic structural properties of 30S subunits and hence of hnRNP. In vitro construction of RNP complexes using model nucleic acid molecules should prove useful to the further study of the processing of mRNA.     

Simultaneous Preparation and Quantitation of Proteoglycans by Precipitation with Alcian Blue

Conditions for specific interaction between Alcian blue and proteoglycans were optimized by comparing the differential spectra of Alcian blue obtained with purified chondroitin sulfate dissolved in water with the spectra obtained with nasal cartilage proteoglycans dissolved in synovial fluid. A method was then designed that provides specific precipitation of proteoglycans or glycosaminoglycans in 4 M guanidine-HCl in the presence of protein, hyaluronic acid, or nucleic acids. The specificity is achieved by using a low pH in combination with detergent and high salt concentration. Stepwise addition of reagents is necessary to avoid binding of Alcian blue to proteins and nucleic acids. All polyanions, except polysulfates, are first neutralized by lowering the pH to 1.5. By including detergent in this step, the hydrophobic protein regions are blocked and not accessible for binding with the dye. These regions could otherwise bind Alcian blue by hydrophobic interaction. When the Alcian blue reagent is added after, only the polysulfated molecules will remain charged and free to interact with Alcian blue. At least 0.4 M guanidine-HCl is required to abolish the negative interference by proteins. All sulfated glycosaminoglycans are precipitated at 0.4 M guanidine-HCl. With increasing guanidine-HCl concentrations, the different glycosaminoglycans are precipitated in accordance with the critical electrolyte concentration of the respective glycosaminoglycan. The Alcian blue precipitation can be performed at different concentrations of guanidine-HCl in order to separate different classes of proteoglycans. Excess dye and contaminating proteins are removed by a wash in a DMSO-MgCl₂ solution and the precipitate is dissolved in a mixture of guanidine-HCl and propanol. For quantitation, the absorbance is recorded in a microplate reader with the 600-nm filter, the assay being linear between 0.5 and 20 μg proteoglycan. Since no digestion of samples with protease is needed, the proteoglycans are recovered in native form. The proteoglycan-Alcian blue complexes dissociate in the guanidine-HCl/propanol mixture and the proteoglycans can be selectivelyprecipitated with propanol. The dye is used for quantitation and the proteoglycans can be utilized for further analysis.     

Modeling protein–nucleic acid complexes with extremely large conformational changes using Flex-LZerD

Proteins and nucleic acids are key components in many processes in living cells, and interactions between proteins and nucleic acids are often crucial pathway components. In many cases, large flexibility of proteins as they interact with nucleic acids is key to their function. To understand the mechanisms of these processes, it is necessary to consider the 3D atomic structures of such protein–nucleic acid complexes. When such structures are not yet experimentally determined, protein docking can be used to computationally generate useful structure models. However, such docking has long had the limitation that the consideration of flexibility is usually limited to small movements or to small structures. We previously developed a method of flexible protein docking which could model ordered proteins which undergo large-scale conformational changes, which we also showed was compatible with nucleic acids. Here, we elaborate on the ability of that pipeline, Flex-LZerD, to model specifically interactions between proteins and nucleic acids, and demonstrate that Flex-LZerD can model more interactions and types of conformational change than previously shown.     

1D2DSimScore: A novel method for comparing contacts in biomacromolecules and their complexes

The biologically relevant structures of proteins and nucleic acids and their complexes are dynamic. They include a combination of regions ranging from rigid structural segments to structural switches to regions that are almost always disordered, which interact with each other in various ways. Comparing conformational changes and variation in contacts between different conformational states is essential to understand the biological functions of proteins, nucleic acids, and their complexes. Here, we describe a new computational tool, 1D2DSimScore, for comparing contacts and contact interfaces in all kinds of macromolecules and macromolecular complexes, including proteins, nucleic acids, and other molecules. 1D2DSimScore can be used to compare structural features of macromolecular models between alternative structures obtained in a particular experiment or to score various predictions against a defined “ideal” reference structure. Comparisons at the level of contacts are particularly useful for flexible molecules, for which comparisons in 3D that require rigid-body superpositions are difficult, and in biological systems where the formation of specific inter-residue contacts is more relevant for the biological function than the maintenance of a specific global 3D structure. Similarity/dissimilarity scores calculated by 1D2DSimScore can be used to complement scores describing 3D structural similarity measures calculated by the existing tools.     

The in vitro DNA-binding properties of purified nuclear lamin proteins and vimentin

The ability of purified nuclear lamin A, lamin B, lamin C, and vimentin from Ehrlich ascites tumor cells to bind nucleic acids was investigated in vitro via a quantitative filter binding assay. At low ionic strength, vimentin bound more nucleic acid than the nuclear lamins and showed a preference for G-containing nucleic acids. Nuclear lamins A and C were quite similar in their binding properties and bound G- and C-containing nucleic acids preferentially. The binding of poly(dT) by the lamins A and C was reduced in competition experiments by both poly(dG) and poly(dC), but not by poly(dA). Lamin B bound only oligo and poly(dG); no other nucleic acids tested were bound or could compete with the binding of oligo(dG). Vimentin, lamin A, and lamin C specifically bound a synthetic oligonucleotide human (vertebrate) telomere model. The Ka for vimentin (2.7 X 10(7) M-1) was approximately 10-fold higher than those for lamin A (2.8 X 10(6) M-1) and lamin C (2.9 X 10(6) M-1). Lamin B did not bind detectable amounts of the telomere model. Washing of lamin A- and lamin C-nucleic acid complexes, formed at low ionic strength, with solutions containing 150 mM KCl resulted in the elution of 30% of bound poly(dG)12-18 and 70% of bound synthetic oligonucleotide telomere model. These results, using purified individual proteins, are in good agreement with data from competition experiments with vimentin but are at odds with data obtained previously using a crude preparation of nuclear matrix proteins containing all three nuclear lamin proteins (Comings, D. E., and Wallack, A. S. (1978) J. Cell Sci. 34, 233-246). The nuclear lamins A and C and vimentin possess nucleic acid-binding properties that might permit their binding to specific base sequences and/or unique DNA structure, such as that observed for the binding of the telomere model. The significance of the higher affinity binding of nucleic acids by the cytoplasmic protein vimentin (compared with the nuclear lamins) remains to be elucidated.     

Model for the irreversible dissociation kinetics of cooperatively bound protein–nucleic acid complexes

We present a quantitative model for the irreversible dissociation kinetics of cooperatively bound nonspecific protein–nucleic acid complexes. The model assumes that the major pathway of dissociation is via singly contiguously bound protein that “peels” off the ends of clusters of bound protein. It should therefore be most applicable for proteins that bind nucleic acids with high cooperativity (w > 10³). Furthermore, the model assumes that no redistribution of bound protein occurs during the time course of the dissociation. Solutions to the rate equations are presented for the entire time course of the dissociation. Under initial conditions such that the nucleic acid is less than fully saturated with protein, a single-exponential decay is predicted (if w is large). However, when the nucleic acid lattice is initially fully saturated, zero-order kinetics, corresponding to a constant rate of protein dissociation, is predicted. The experimental observation of zero-order dissociation kinetics in a cooperative protein–nucleic acid system is a good qualitative indicator for the dissociation mechanism discussed here. A discussion of the analysis of experimental data that enables one to extract molecular rate constants is presented. Furthermore, comparisons are made between the nonredistributing model presented here and Epstein's model [Epstein, I. R. (1979) Biopolymers 18 , 2037–2050] in which protein can translocate infinitely quickly while bound to the nucleic acid, and hence protein clusters redistribute during dissociation and maintain an equilibrium distribution on the nucleic acid at all times.     

Participation of manganese ions complexed with tRNA in the interaction with amino acids and dipeptides.

Carboxyl groups of glycine, beta-alanine, gamma-aminobutyric acid and diglycine interact with Mn2+ coordinated by tRNA, as revealed by 1H and 13C NMR studies. The amino groups of these compounds interact with tRNA phosphate groups. The distances between the coordinated Mn2+ and carboxyl groups and the alpha-protons of glycine were determined. The role of Mn2+ and Mg2+ complexed with nucleic acids in the formation of specific complexes between proteins and nucleic acids is discussed.