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.     

Cross-linking of elongation factor 2 to rat-liver ribosomal proteins by 2-iminothiolane

Complexes containing rat liver 80S ribosomes treated with puromycin and high concentrations of KCl, elongation factor 2 (EF-2) from pig liver, and guanosine 5'-[beta, gamma-methylene]triphosphate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 22 fractions by chromatography on carboxymethylcellulose of which seven fractions were used for further analyses. Each protein fraction was subjected to diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Nine cross-linked protein pairs between EF-2 and ribosomal proteins were shifted from the line formed with monomeric proteins. The spots of ribosomal proteins cross-linked to EF-2 were cut out from the gel plate and labelled with 125I. The labelled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both large and small subunits were identified: L9, L12, L23, LA33 (acidic protein of Mr 33000), P2, S6 and S23/S24, and L3 and L4 in lower yields. The results are discussed in relation to the topographies of ribosomal proteins in large and small subunits. Furthermore we found new neighboring protein pairs in large subunits, LA33-L11 and LA33-L12.     

Neighboring proteins in rat liver 60 S ribosomal subunits disulfide-linked by hydrogen peroxide oxidation or cross-linked with dithiobis(succinimidyl propionate)

Neighboring proteins in rat liver 60 S ribosomal subunits were investigated by two kinds of cross-linking techniques: treatment of 60 S subunits with 1) hydrogen peroxide, which promotes the formation of protein-protein disulfide linkages and 2) a disulfide-bridged bifunctional reagent dithiobis(succinimidyl propionate). The cross-linked protein complexes formed were separated by two-dimensional polyacrylamide gel electrophoresis in a basic-sodium dodecyl sulfate gel system under nonreducing conditions. Each complex in the gel was labeled with 125I and extracted under reducing conditions. The protein components of the complex were analyzed by two kinds of two-dimensional polyacrylamide gel electrophoresis, followed by autoradiography. Closely neighboring pairs disulfide-linked by hydrogen peroxide were identified as L4-L6, L4-L29, L6-L29, L18a-L29, and L29-L32; more distant pairs cross-linked with dithiobis(succinimidyl propionate) were identified as L3-L5, L3-L24, L3-L37a, L4-L14, L4-L18a, L5-L10, L5-L11, L7/L7a-L27, L7/L7a-L36, L13-L35, and L13a-L14.     

Cross-linking study on protein neighborhoods at the subunit interface of rat liver ribosomes with 2-iminothiolane

Rat liver 80 S ribosomes were cross-linked with 2-iminothiolane. Proteins extracted from the cross-linked 80 S ribosomes were separated into 25 fractions by chromatography on carboxy methylcellulose. Each protein fraction was analyzed by diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Eight pairs characteristic of 80 S ribosomes were detected which did not appear when isolated 40 S and 60 S subunits were cross-linked, and the cross-linked proteins were analyzed in similar manners. The cross-linked components were radioiodinated and then analyzed by two-dimensional gel electrophoresis, followed by autoradiography. Eight kinds of cross-links between 60 S subunit proteins and 40 S subunit proteins were identified as follows: SA30 (acidic protein with Mr 30,000)-LA33 (acidic protein with Mr 33,000), S2-LA33, S2-L11, S3a-L11, S4-L5, S25-L5, S4-L24 and S6-L24.     

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.     

Extensive cross-linking of calf brain plain synaptic vesicle proteins

Calf brain plain synaptic vesicle proteins have been cross-linked with bis[2-(succinimidooxycarbonyloxy)ethyl] sulfone, a homobifunctional, cleavable reagent, as well as with N-hydroxysuccinimidyl 4-azidobenzoate, a photosensitive, heterobifunctional reagent. These results demonstrate the generality of a recent report that synaptic vesicle proteins can be cross-linked, in contrast to a prior report that no cross-linking could be observed. The reagents gave some differences in the proteins that were preferentially cross-linked. A protein at Mr = 173 000, which comigrates with clathrin, is present in the plain synaptic vesicle fraction and appears to be involved in cross-linking. A high degree of association and structural organization of synaptic vesicle proteins is suspected, since extensive cross-linking of most synaptic vesicle proteins with high-molecular-mass proteins, which are probably structural in nature, is observed. A protein with an Mr of 249 000 is specifically cross-linked to a protein of Mr 42 000, probably actin, suggesting that the 249 000-Mr protein may be a spectrin-like molecule. The present results suggest that synaptic vesicles may be organized by a spectrin-like matrix similar to that observed in erythrocytes and other cells.     

Topography of the C. coli 5S RNA-protein complex as determined by crosslinking with dimethyl suberimidate and dimethyl-3,3''-dithiobispropionimidate

5S RNA-protein complexes were prepared in vitro using partially purified E. coli 5S RNA and total E. coli 70S ribosomal proteins. The complexes were isolated from sucrose gradients and shown to contain proteins L5, L18, L25 and a fourth protein not heretofore characterized and designed L31. The complexes were treated with the crosslinking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate. Both reagents gave identical patterns of crosslinked proteins when analyzed by one-dimensional polyacrylamide/dodecylsulfate gel electrophoresis. Dimers of L5-L31', L5-L18 and L18-L18 and a trimer containing L5, L18 and L31' were identified by diagonal polyacrylamide/dodecylsulfate gel electrophoresis of the proteins crosslinked with dimethyl-3,3'-dithiobispropionimidate. No crosslinking was detected between L25 and the other three proteins.     

Identification by RNA-protein cross-linking of ribosomal proteins located at the interface between the small and the large subunits of mammalian ribosomes

Protein constituents at the subunit interface of rat liver ribosomes were analysed by cross-linking with the bifunctional reagent, diepoxybutane (distance between reactive groups 4 A). Isolated 40S and 60S subunits were labelled with 125I and recombined with unlabelled complementary subunits. The two kinds of selectively labelled 80S ribosomes were treated with diepoxybutane at low concentration. Radioactive ribosomal proteins covalently attached to the rRNA of the unlabelled complementary subparticles were isolated by repeated gradient centrifugation. The RNA-bound, labelled proteins were identified by two-dimensional gel electrophoresis. The experiments showed that proteins S2, S3, S4, S6, S7, S13, and S14 in the small subunit of rat liver ribosomes are located at the ribosomal interface in close proximity to 28S rRNA. Similarly, proteins L3, L6, L7, and L8 were found at the the interface of the large ribosomal subunit in the close vicinity of 18S rRNA.     

Ribosomal protein L7/L12 cross-links to proteins in separate regions of the 50 S ribosomal subunit of Escherichia coli

The 50 S ribosomal subunits from Escherichia coli were modified by reaction with 2-iminothiolane under conditions in which 65 sulfhydryl groups, about 2/protein, were added per subunit. Earlier work showed that protein L7/L12 was modified more extensively than the average but that nearly all 50 S proteins contained sulfhydryl groups. Mild oxidation led to the formation of disulfide protein-protein cross-links. These were fractionated by urea gel electrophoresis and then analyzed by diagonal gel electrophoresis. Cross-linked complexes containing two, three, and possibly four copies of L7/L12 were evident. Cross-links between L7/L12 and other ribosomal proteins were also formed. These proteins were identified as L5, L6, L10, L11, and, in lower yield, L9, L14, and L17. The yields of cross-links to L5, L6, L10, and L11 were comparable to the most abundant cross-links formed. Similar experiments were performed with 70 S ribosomes. Protein L7/L12 in 70 S ribosomes was cross-linked to proteins L6, L10, and L11. The strong L7/L12-L5 cross-link found in 50 S subunits was absent in 70 S ribosomes. No cross-links between 30 S proteins and L7/L12 were observed.     

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.     

Cross-linking study on localization of the binding site for elongation factor 1 alpha on rat liver ribosomes

Complexes containing rat liver 80 S ribosomes, poly(uridylic acid), phenylalanyl-tRNA, elongation factor 1 alpha, and guanylyl(beta, gamma-methylene)-diphosphonate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 26 fractions by chromatography on carboxymethylcellulose. Each protein fraction was subjected to diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Four cross-linked pairs containing elongation factor 1 alpha were on the vertical line below the diagonal. The ribosomal protein spot of each pair was cut out from the gel plate and labeled with 125I. The labeled proteins were extracted from the gel and identified by two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both 60 S and 40 S subunits were identified: L12, L23, L39, S23/S24, and S26, three proteins of which had been found to be cross-linked also to elongation factor 2 (Uchiumi, T., Kikuchi, M., Terao, K., Iwasaki, K., and Ogata, K. (1986) Eur. J. Biochem. 156, 37-44). These results afford direct evidence that both elongation factors interact with partially overlapping sites on rat liver ribosomes.     

Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli

We have carried out an extensive protein-protein cross-linking study on the 50S ribosomal subunit of Escherichia coli using four different cross-linking reagents of varying length and specificity. For the unambiguous identification of the members of the cross-linked protein complexes, immunoblotting techniques using antisera specific for each individual ribosomal protein have been used, and for each cross-link, the cross-linking yield has been determined. With the smallest cross-linking reagent diepoxybutane (4 A), four cross-links have been identified, namely, L3-L19, L10-L11, L13-L21, and L14-L19. With the sulfhydryl-specific cross-linking reagent o-phenylenedimaleimide (5.2 A) and p-phenylenedimaleimide (12 A), the cross-links L2-L9, L3-L13, L3-L19, L9-L28, L13-L20, L14-L19, L16-L27, L17-L32, and L20-L21 were formed; in addition, the cross-link L23-L29 was exclusively found with the shorter o-phenylenedimaleimide. The cross-links obtained with dithiobis(succinimidyl propionate) (12 A) were L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L32, L19-L25, L20-L21, and L23-L34. The good agreement of the cross-links obtained with the different cross-linking reagents used in this study demonstrates the reliability of our cross-linking approach. Incorporation of our cross-linking results into the three-dimensional model of the 50S ribosomal subunit derived from immunoelectron microscopy yields the locations for 29 of the 33 proteins within the larger ribosomal subunit.     

Disulfide interaction in situ between two neighbouring proteins in mammalian 60-S ribosomal subunits. Isolation of the contact region of the larger protein.

A disulfide complex is formed in situ under gentle conditions between two neighbouring proteins in the 60-S subunits of mammalian ribosomes. The proteins have been identified as L 4 and L 29. The complex is easily isolated from whole ribosomes, and can be utilized for preparing the two proteins in a very pure state for further characterization. Chymotryptic cleavage of the complex or the isolated larger protein (L 4) in the presence of SDS produces two unequal fragments of this protein in nearly quantitative yield. The smaller fragment (approx. 12 000 daltons) contains the contact sequence. Only this fragment of protein L 4 is labelled when rat liver ribosomes are incuabted with iodo[14C]acetate under conditions of complex formation. Protein L 29 is resistant to chymotrypsin in the presence of sodium dodecyl sulfate.     

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.     

Synthesis and degradation in vivo of a phosphoprotein from rat dental enamel. Identification of a phosphorylated precursor protein in the extracellular organic matrix.

The cellular enamel organ and the cell-free organic matrix of developing enamel of female rats injected intravascularly with [3H]serine and [3H]proline were extracted in a number of solvents and examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and h.p.l.c. in 6M-guanidinium chloride at intervals varying from 5 min to 1 week after injection. Three major species soluble in NH4HCO3 with Mr values of approx. 100 000, 25 000 and 11 000 were identified in the cellular enamel organ. The Mr 100 000 and 11 000 components were not secreted but remained intracellular for periods of up to 1 week after injection of the radioactively labelled amino acids. In contrast, the Mr 25 000 species was secreted from the cells and was first detected in the extracellular organic matrix approx. 15-30 min after injection. With time, labelled components, first of Mr approx. 11 000 and subsequently approx. 6500, were detected in the organic matrix concomitant with a relative decrease in the Mr 25 000 component, demonstrating that the lower Mr species were derived from degradation of the putative extracellular precursor protein (Mr 25 000). All of the extracellular components were found to contain O-phosphoserine. No radioactively labelled component with an Mr greater than approx. 25 000, either an amelogenin or an enamelin, was observed in the extracellular organic matrix or in an intracellular component which subsequently was lost from the intracellular pool. The Mr of the highest Mr protein or class of proteins is calculated to be approx. 22 000-26 000 when standard proteins are used as markers, but only 15 000-18 000 when using the CNBr peptides of alpha 1 chains of rat tail tendon collagen as markers.     

Identification of the immunoglobulin G receptor of human platelets

The binding site of IgG on human platelets was studied by the use of the cleavable heterobifunctional cross-linking agent N-succinimidyl (4-azidophenyldithio)propionate. Binding characteristics of the derivatized IgG were similar to normal IgG. Periodate-borohydride treatment of platelets also did not significantly alter their ability to bind IgG. N-Succinimidyl (4-azidophenyldithio)propionate was bound to IgG via a succinimidyl ester and then photolyzed in the presence of intact platelets. Their membrane glycoproteins were first tritiated by the periodate-borohydride method. The cross-linked product was analyzed by two dimensional sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The non-reduced first-dimension gels were subjected to 5% 2-mercaptoethanol prior to separation in the second dimension. Such gels were then evaluated by fluorography, silver staining, and counting the radioactivity of sequential gel strips in the area of cross-linking. The protein complexes at the interface between stacking and running gel were further resolved in isoelectric focusing gels. One IgG-containing band could be identified. After reduction, the constituent proteins of the cross-linked complex were analyzed by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and subsequent immunoblotting with an antiserum against platelet membrane glycoproteins. All of these studies gave evidence of glycoprotein IIIa as the receptor of IgG. Based on the results of the different experimental approaches, we conclude that glycoprotein IIIa is the IgG receptor in human platelets.     

Affinity-labelling of the thyrotropin receptor. Characterization of the photoactive ligand.

Thyrotropin (TSH) has been coupled to the photoactive heterobifunctional reagent N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) and the properties of the product (HSAB-TSH) investigated. Preparations of HSAB-TSH containing two molecules of HSAB per molecule of TSH were used in most experiments and these preparations retained about 40% of the original receptor-binding activity of the TSH. HSAB-TSH could be labelled with 125I and cross-linked to porcine and human TSH receptors. Analysis of the cross-linked complexes indicated that the receptors consisted of two subunits (designated A and B) linked by a disulphide bridge. In the case of the human TSH receptor, the A- and B-subunits had approximate Mr values of 50 000 and 30 000 respectively, whereas the Mr values for porcine TSH-receptor A- and B-subunits were approx. 45 000 and 25 000 respectively. Only the A subunit was cross-linked to TSH. Comparison of the effects of trypsin and mercaptoethanol on the TSH-TSH-receptor complexes suggested that the trypsin cleavage point on the A-subunit was at a point close to the disulphide bridge.     

Heterobifunctional cross-linking of a monoclonal antibody with 2-methyl-N1-benzenesulfonyl-N4-bromoacetylquinonediimide

The Cyssor reagent, 2-methyl-N1-benzenesulfonyl-N4-bromoacetylquinonediimide, which will cleave a protein chain at Cys under acidic conditions, cross-linked unreduced and partially reduced antibody at pH 8.0. No cleavage of the antibody occurred suggesting that the Cyssor reagent may be useful with certain proteins as a heterobifunctional cross-linker.     

Location of the sulfhydryl groups involved in disulfide interaction between the neighboring proteins L6 and L29 in mammalian ribosomes. S-cleavage of the cyanylated proteins in polyacrylamide gels after separation by dodecylsulfate gel electrophoresis

Proteins L6 and L29 occupy closely adjacent sites in mammalian 60-S ribosomal subparticles and are easily cross-linked by intermolecular disulfide bond formation. For locating the interacting thiols within the polypeptide chains the dissociated proteins L6 and L29 obtained from the isolated disulfide complex were subjected to S-cleavage following [14C]cyanylation of the two cysteine residues. Four split products of the [14C]cyanylated proteins were isolated by dodecylsulfate gel electrophoresis. Two of these could be identified by autoradiography as the selectively labeled C-terminal fragments. For unequivocal assignment of the fragments to the parent proteins, a simple and generally applicable method of cleaving cyanylated proteins in polyacrylamide gel for subsequent diagonal analysis was developed. The experiments indicated that the sulfhydryl group of L6 interacting with L29 is located at a distance of approximately 80 amino acid residues from the N-terminus. In the intact ribosome this sequence contains a clostripain-sensitive and trypsin-sensitive portion of the protein more or less exposed at the ribosomal surface. In the case of protein L29, the interacting sulfhydryl group was located at a distance of approximately 40 amino acid residues from the C-terminal.     

Controlled coupling of mildly reduced proteins to Sepharose gelatin by heterobifunctional reagent

The heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate was utilized for controlled coupling of mildly reduced BSA and lysozyme to Sepharose gelatin to prepare immunoabsorbents. Each reaction step was examined and quantitated. The free amino groups on gelatin after coupling gelatin to cyanogen bromide-activated Sepharose as well as the number of 3-(2-pyridyldithio) propionyl groups on Sepharose gelatin were quantitated. Coupling of mildly reduced BSA as well as lysozyme to PDP-Sepharose gelatin occurred through sulfhydryl-disulfide exchange and permitted the formation of an immunoabsorbent. The immunoabsorbents were capable of binding the respective antibody and the eluted antibodies were pure and free of plasma proteins.