No abstract

The bifunctional reagent, methyl 4-mercaptobutyrimidate, has been useful in identifying neighboring protein pairs in the Escherichia coli 30S ribosomal subunit. The reagent reacts with protein amino groups and upon oxidation forms intermolecular disulfide bonds. The compound has been characterized further employing the techniques of nuclear magnetic resonance spectroscopy, infrared spectroscopy, ultraviolet spectroscopy, mass spectroscopy, elemental analysis, and sulfhydryl group titration. All of the results indicate that the compound formerly considered to be methyl 4-mercaptobutyrimidate is in fact 2-iminothiolane (2-iminotetrahydrothiophene). Unlike most imidates, 2-iminothiolane is highly stable in solution at acidic and neutral pH. Furthermore, we have not observed any protein:protein cross-links produced withThe bifunctional reagent methyl 4-mercaptobutyrimidate, used previously in this laboratory to cross-link neighboring ribosomal proteins, has been found to have properties indicating its identity with the compound 2-iminothiolane described by Schramm & Diilffer [Schramm, H. J., & Diilffer, T. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358,[137][138][139], The latter name will be used here.

Antibodies with bound metal-chelate haptens provide new means for exploiting the diverse properties of metallic elements. The murine monoclonal antibody CHA255 (IgG1 lambda) binds the metal-chelate hapten indium (III)-4-[N'-(2-hydroxyethyl)thioureido]-L-benzyl-EDTA (designated In-EOTUBE) with high affinity (K(a) = 1.1 x 10(10) M-1). Antibody binding is highly specific for the indium chelate; the affinity decreases as much as 10(4) with other metals, even those having ionic radii close to indium. To better understand this selectivity, the crystal structure of the antigen-binding fragment (Fab') of CHA255 complexed with its hapten, In(III)-EOTUBE, was determined by molecular replacement and refined at 2.2-A resolution. The structure of CHA255 Fab' complexed with Fe(III)-EOTUBE was also determined and refined at 2.8-A resolution. In both structures, the hapten's EDTA moiety is half-buried near the center of the complementarity-determining regions (CDR's). Five of the six CDR's on the Fab' interact with the hapten through protein side-chain atoms (but not main-chain atoms). A novel feature of the In-EOTUBE/Fab' complex is coordination of the indium by N epsilon of one histidine from the heavy chain's third CDR (distance = 2.4 A). The histidine coordination is not observed in the Fe-EOTUBE/Fab' complex, due mainly to a slightly different hapten conformation that reduces metal accessibility; this may partially explain the 20-fold lower affinity of CHA255 for iron hapten. An unexpected feature of the Fab' overall is an elbow angle of 193 degrees (the angle between the pseudodyad axes of the Fab's constant and variable domains).

An antibody (IgG1) was designed for oriented adherence to a metal-containing surface. This was achieved by adding a metal-chelating peptide, (CP = His-Trp-His-His-His-Pro), to the COOH-terminus of the heavy chain through genetic engineering. Electroporation of the engineered heavy chain gene into cells expressing the complimentary light chain yielded colonies secreting an intact antibody containing the metal-chelating peptide (IgG1-CP) which had high affinity for a nickel-loaded iminodiacetate column. Purified IgG1-CP was bound to nickel-treated mica and imaged by atomic force microscopy (AFM). Antibody lacking the COOH-terminal metal binding peptide failed to produce discernible AFM images. The AFM images of individual IgG1-CP molecules and their calculated dimensions demonstrated that regiospecific binding and uniform orientation of the antibody was imparted by the peptide. The ability to stably orient macromolecules in their native state to a surface may be used advantageously to visualize them.

The 3 0 s ribosomal subunits of Escherichia coli were modified with 2-iminothiolane with the formation of amidine-linked sulfhydryl groups attached to ribosomal protein. The modified particle contained 61 sulfhydryl groups, 17-18 due to endogenous cysteine residues and the remainder from modification. The modified ribosomal subunits were oxidized to promote disulfide bond formation (cross-linking). About 15 free sulfhydryl groups per 3 0 s particle remained after oxidation even when performed in the presence of 2mercaptoethanol. Treatment of modified, oxidized particles with 4.0 M urea, 3.0 M LiCl exposed these sulfhydryl groups which reacted with iodoacetamide only after disruption of the native structure. The presence of these sulfhydryl groups prompted an investigation of possible sulfhydryl/disulfide interchange and random oxidation during extraction of cross-linked ribosomal proteins and/or the preparation of protein for diagonal polyacrylamide/dodecyl sulfate gel electrophoresis. Experiments were carried out to obtain direct evidence concerning the quantitative contribution of disulfide interchange and/or random oxidation during protein extraction to the pattern of cross-linked dimers previously reported. A radiolabeled cross-linked protein fraction of about 35 500 molecular weight was purified from cross-linked 35S-labeled 3 0 s subunits. The radiolabeled protein was added to nonra-C h e m i c a l cross-linking of biological structures containing several polypeptide chains can provide useful information on the topography of the protein constituents. Such evidence is particularly valuable in the study of molecular structures for which X-ray crystallographic evidence is lacking or inadequate (Petlrs & Richards, 1977). A fundamental requirement is that From the