Synthesis and properties of a new cleavable nucleic acid-protein crosslinking reagent

A new compound, dithiobis[9-(2-ethylenecarbamoylethylamino)-2,3-dimethoxy-6-azido-acridine], was synthesized and used in some preliminary experiments to form cleavable complexes between nucleic acids and proteins. In a first step the azidoacridine moiety of the reagent intercalates between the bases of nucleic acids and is then bound by reaction of the azido group. The disulfide group of the reagent is simultaneously converted under reducing conditions into a thiol which, in a second step, can be bound by oxidation to -SH groups of a vicinal protein (additional -SH groups can be inserted in the protein using 2-iminothiolane). The nucleic acid-protein complexes thus formed can be redissociated by reduction. The potential applications of this new cleavable crosslinking reagent could be extended to topographical investigations of any biological structure composed of nucleic acids and proteins.     

A new cleavable reagent for cross-linking and reversible immobilization of proteins.

We have prepared a new bifunctional reagent for the cross-linking and reversible immobilization of proteins through their amine groups. This compound, ethylene glycolyl bis(succinimidyl succinate), reacts rapidly with proteins, at pH 7 and at high dilution. The resulting protein cross-links are readily cleaved at pH 8.5 using hydroxylamine for 3–6 hr. at 37°C. Substantial enzymatic activity was observed with lactic dehydrogenase after such reversible cross-linking. Trypsin immobilized on agarose using this reagent retains full specific activity, is stable for weeks in the cold, and may be released with hydroxylamine at 25°C. This compound appears suitable for studies involving proteins with essential disulfide linkages.     

A 125I-radiolabel transfer crosslinking reagent with a novel cleavable group

A new chemical crosslinking reagent, 1-[N-(2-hydroxy-5-azidobenzoyl) -2-aminoethyl]-4-(N-hydroxysuccinimidyl)-succinate, or HAHS, has been developed. It is synthesized in three steps and stored as an unlabeled precursor, and then iodinated immediately before use. The reagent has a succinimide ester at one end so that it can be covalently attached to a purified protein, and a radioiodinated phenyl azide group at the other end, so that upon photolysis it can form crosslinks to nearby molecules. The 16-A connecting region contains an ester group which is very stable at neutral pH before photolysis, but which hydrolyzes in about 1 min in base, and hydrolyzes spontaneously after photolysis. Thus, photolysis and cleavage of the ester result in transfer of the radiolabel from the initial protein to its neighbors. When 125I-HAHS-protein A was incubated with IgG, photolyzed, and cleaved, 27% of the label was transferred to the IgG heavy chain. This transfer was abolished by an excess of unlabeled protein A, and was quenched by low concentrations of DTT. Much lower amounts of label were transferred to noninteracting proteins. When 125I-HAHS-spectrin was bound to spectrin-depleted red blood cell membranes, photolyzed, and cleaved, label was transferred only to ankyrin and to band 3. This transfer was blocked by excess unlabeled spectrin and was greatly diminished by conditions which prevent binding of spectrin.     

Three-dimensional structure of the yeast ribosome.

The 80S ribosome from Saccharomyces cerevisiae has been reconstructed from cryo electron micrographs to a resolution of 35 A. It is strikingly similar to the 70S ribosome from Escherichia coli, while displaying the characteristic eukaryotic features familiar from reconstructions of ribosomes from higher eukaryotes. Aside from the elaboration of a number of peripherally located features on the two subunits and greater overall size, the largest difference between the yeast and E.coli ribosomes is in a mass increase on one side of the large (60S) subunit. It thus appears more elliptical than the characteristically globular 50S subunit from E.coli. The interior of the 60S subunit reveals a variable diameter tunnel spanning the subunit between the interface canyon and a site on the lower back of the subunit, presumably the exit site through which the nascent polypeptide chain emerges from the ribosome.     

Reversible inactivation of (Na+ + K+)-ATPase by use of a cleavable bifunctional reagent.

1. Purified (Na+ + K+)-ATPase, prepared from rabbit kidney outer medulla, is incubated with the bifunctional NH2-directed reagent dimethyl 3,3'-dithiobis-propionimidate. This results in a cross-link between the subunits of the enzyme and a simultaneous reduction of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 2. The most abundant cross-link product is a dimer of the two different subunits of the enzyme. 3. Reduction of the disulfide cross-link by dithioerythritol results in partial recovery of the original subunit structure of the enzyme and of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 4. These results suggest that a free mobility of the subunits of the (Na+ + K+)-ATPase system relative to each other is essential for proper functioning of both enzyme activities.     

Crystal structure of the ribosome recycling factor bound to the ribosome

In bacteria, disassembly of the ribosome at the end of translation is facilitated by an essential protein factor termed ribosome recycling factor (RRF), which works in concert with elongation factor G. Here we describe the crystal structure of the Thermus thermophilus RRF bound to a 70S ribosomal complex containing a stop codon in the A site, a transfer RNA anticodon stem-loop in the P site and tRNA(fMet) in the E site. The work demonstrates that structures of translation factors bound to 70S ribosomes can be determined at reasonably high resolution. Contrary to earlier reports, we did not observe any RRF-induced changes in bridges connecting the two subunits. This suggests that such changes are not a direct requirement for or consequence of RRF binding but possibly arise from the subsequent stabilization of a hybrid state of the ribosome.     

Synthesis of a water-soluble protein cross-linking reagent

2,4-Difluoro-5-nitrobenzenesulfonic acid has been synthesized by the sulfonation of 2,4-difluoronitrobenzene, and precipitated with KCl as the potassium sulfonate. The structure was confirmed by chemical and spectroscopic methods (IR, 1H-NMR, 13C-NMR, 19F-NMR, UV, MS and ultimate organic analysis). Lysozyme was cross-linked with the potassium sulfonate and with 1,5-difluoro-2,4-dinitrobenzene. The products were analysed by SDS-PAGE and compared. The cross-linking conditions were optimized.     

Three-dimensional structure of the mammalian cytoplasmic ribosome

A three-dimensional reconstruction of the 80 S ribosome from rabbit reticulocytes has been calculated from low-dose electron micrographs of a negatively stained single-particle specimen. At 37 A resolution, the precise orientations of the 40 S and 60 S subunits within the monosome can be discerned. The translational domain centered on the upper portion of the subunit/subunit interface is quite open, allowing considerable space between the subunits for interactions with the non-ribosomal macromolecules involved in protein synthesis. Further, the cytosolic side of the monosome is strikingly more open than the membrane-attachment side, suggesting a greater ease of communication with the cytoplasm, which would facilitate the inwards and outwards diffusion of a number of ligands. Although the 60 S subunit portion of the 80 S structure shows essentially all of the major morphological features identified for the eubacterial 50 S large subunit, it appears to possess a region of additional mass that evidently accounts for the more ellipsoidal form of the eukaryotic subunit.     

Synthesis and characterization of N-(4-azidophenylthio)phthalimide: A cleavable, photoactivable crosslinking reagent that reacts with sulfhydryl groups

The cleavable, photoaffinity label precursor, N-(4-azidophenylthio)phthalimide has been synthesized and purified. The recrystallized product was identified by infrared spectroscopy and nuclear magnetic resonance spectroscopy. The compound modifies free thiol groups to yield the corresponding S-4-azidophenylthio derivatives. In order to examine the biological applications of this compound, yeast iso-1-cytochrome c, containing a single free cysteine residue, was modified and characterized. The 102-S-(4-azidophenylthio)-iso-1-cytochrome c was found to contain 1 mol of label/mol of cytochrome c. The photoaffinity labeling of purified, detergent-solubilized yeast cytochrome c oxidase was examined. Photolysis products of crosslinking could be analyzed on sodium dodecyl sulfate-polyacrylamide gels in the absence of reducing agents. The crosslinked products were readily cleaved by dithiothreitol. The use of this compound as a sulfhydryl-specific photolabile, bifunctional crosslinking reagent is discussed.     

Chemical probes of extended biological structures: Synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate)

The synthesis and properties of a new cleavable protein cross-linking reagent, [³⁵S]dithiobis(succinimidyl propionate), are detailed. Free primary and secondary aliphatic amino groups are quantitatively acylated by the reagent in either organic or aqueous media within two minutes at 23 °C. By contrast, the half-time for hydrolysis of the active ester termini in buffer at pH 7 is four to five hours, so that protein cross-linkage can be optimized by application of low concentrations of reagent. Accessible amino groups of hemoglobin are acylated with extreme rapidity of 0 °C in pH 7 buffer when [³⁵S]dithiobis(succinimidyl propionate) is applied in 0.4 to 9-fold molar excess. Submicrogram quantities of the cross-linked hemoglobin subunits which result are detectable by monitoring the ³⁵S distribution in sodium dodecyl sulfate-polyacrylamide gels. In addition to amine acylation, two of the six thiol groups in hemoglobin, tentatively located at cysteine 93 of the β chains, are reversibly modified at 0 °C by mercaptan-disul-fide interchange with the reagent or its bis amide analogs. This equilibrium-controlled, pH-dependent reaction occurs at a slower rate than acylation, and is blocked by short preincubation of the protein with N-ethylmaleimide or by addition of 3,3′- dithiodipropionamide (or other disulfides) to the reaction mixture. Disulfides introduced into hemoglobin by acylation and interchange are quantitatively cleaved by reduction for 30 minutes at 37 °C with 10 mm-dithioerythritol buffered at pH 8.5.The properties of high reactivity under mild conditions, long solution half-life, and the radioactive label make [³⁵S]dithiobis(succinimidyl propionate) a particularly useful and versatile probe of extended structures in a variety of biological systems.     

The structure of ribosome decoding center

To develop the hypothesis of direct interaction between the ribosome and the codon-anticodon sugar-phosphate backbones the model of the ribosome decoding centre structure is proposed. The model is based on the structural complementarity between the protein beta-sheet and the narrow groove of double-stranded RNA. A right handed two-stranded antiparallel beta-sheet is the main structural element of the model. One peptide strand interacts with the codon sugar-phosphate backbone while the second interacts with the anti-codon sugar-phosphate backbone. These stereoregular structures interact between them forming a regular system of H-bonds. The model postulates a specific rule of repetition for some amino acids in both peptide strands. Analysis of known sequences of the E. coli ribosomal proteins indicates such boxes of repeated arginine in proteins S1, S10, S11, S14 and boxes of repeated glycine in proteins S5, S11, (L15. These proteins are discussed as candidates for forming the corresponding "arginine" or "glycine" beta-sheets of the decoding centre and interaction with the codon-anticodon pairs. The properties of such interactions are analysed as well.     

The monothioacetal group, a new cleavable center for cross-linking reagents.

A protein cross-linking reagent which contains a monothioacetal moiety is described. Cross-links generated using this reagent may be specifically cleaved by dilute mercuric ion at neutral pH.