Natural poly-histidine affinity tag for purification of recombinant proteins on cobalt(II)-carboxymethylaspartate crosslinked agarose.

A natural 19-amino-acid poly-histidine affinity tag was cloned at the N-terminus of three recombinant proteins. The vectors containing the DNA of the fusion proteins were used for transformation of Escherichia coli DH5alpha cells. Each protein was expressed, extracted and purified in one chromatographic step. The purification procedure for each protein can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent--Co2+-carboxymethylaspartate agarose Superflow--was utilized at linear flow-rates as high as 5 cm/min. The final preparation of each protein is with purity greater than 95% as ascertained by sodium dodecyl sulfate-electrophoresis. Recovery for each purified protein was higher than 77% of the initial loaded amount as judged by biological activity. The operational capacity of Co2+-carboxymethylaspartate agarose for each protein was determined.

A single-headed dimer of Escherichia coli ribosomal protein L7/L12 supports protein synthesis.

During protein synthesis, the two elongation factors Tu and G alternately bind to the 50S ribosomal subunit at a site of which the protein L7/L12 is an essential component. L7/L12 is present in each 50S subunit in four copies organized as two dimers. Each dimer consists of distinct domains: a single N-terminal ("tail") domain that is responsible for both dimerization and binding to the ribosome via interaction with the protein L10 and two independent globular C-terminal domains ("heads") that are required for binding of elongation factors to ribosomes. The two heads are connected by flexible hinge sequences to the N-terminal domain. Important questions concerning the mechanism by which L7/L12 interacts with elongation factors are posed by us in response to the presence of two dimers, two heads per dimer, and their dynamic, mobile properties. In an attempt to answer these questions, we constructed a single-headed dimer of L7/L12 by using recombinant DNA techniques and chemical cross-linking. This chimeric molecule was added to inactive core particles lacking wild-type L7/L12 and shown to restore activity to a level approaching that of wild-type two-headed L7/L12.

Cross-linking of selected residues in the N- and C-terminal domains of Escherichia coli protein L7/L12 to other ribosomal proteins and the effect of elongation factor Tu.

Five different variants of protein L7/L12, each with a single cysteine substitution at a selected site, were produced, modified with 125I-N-[4-(p-azidosalicylamido)-butyl]-3-(2'-pyridyldithio)propion amide, a radiolabeled, sulfhydryl-specific, heterobifunctional, cleavable photocross-linking reagent that transfers radiolabel to the target molecule upon reduction of the disulfide bond. The proteins were reconstituted with core particles depleted of wild type L7/L12 to yield 70 S ribosomes. Cross-linked molecules were identified and quantified by the radiolabel. No cross-linking of RNA was detected. Two sites in the dimeric N-terminal domain, Cys-12 and Cys-33, cross-linked strongly to L10 and in lower yield to L11 but to no other proteins. The three sites in the globular C-terminal domain all cross-linked strongly to L11 and, in lower yield, to L10. Weaker cross-linking to 50 S proteins L2 and L5 occurred from all three C-terminal domain locations. The 30 S ribosomal proteins S2, S3, S7, S14, S18 were also cross-linked from all three of these sites. Binding of the ternary complex [14C]Phe-tRNA-elongation factor Tu.guanyl-5'-yl imidodiphosphate) but not [14C]Phe-tRNA.elongation factor Tu.GDP.kirromycin increased labeling of L2, L5, and all of the 30 S proteins. These results imply the flexibility of L7/L12 and the transient proximity of three surfaces of the C-terminal domain with the base of the stalk, the peptidyl transferase domain, and the head of the 30 S subunit.

Rotational and conformational dynamics of Escherichia coli ribosomal protein L7/L12.

Fluorescence methods were utilized to study dynamic aspects of the 24 kDa dimeric Escherichia coli ribosomal protein L7/L12. Oligonucleotide site-directed mutagenesis was used to introduce cysteine residues at specific locations along the peptide chain, in both the C-terminal and N-terminal domains, and various sulfhydryl reactive fluorescence probes (iodoacetamido) fluorescein, IAEDANS, pyrenemethyl iodoacetate) were attached to these residues. In addition to the full-length proteins, a hinge-deleted variant and variants corresponding to the C-terminal fragment and the N-terminal fragment were also studied. Both steady-state and time-resolved fluorescence measurements were carried out, and the results demonstrated that L7/L12 is not a rigid molecule. Specifically, the two C-terminal domains move freely with respect to one another and with respect to the dimeric N-terminal domain. Removal of the hinge region, however, significantly reduces the mobility of the C-terminal domains. The data also show that the rotational relaxation time monitored by the fluorescent probe-depends upon the probe's excited state lifetime. This observation is interpreted to indicate that a hierarchy of motions exists in the L7/L12 molecule including facile motions of the C-terminal domains and dimeric N-terminal domain, in addition to the overall tumbling of the protein. Probes attached to the N-terminal domain exhibit global rotational relaxation times consistent with the molecular mass of the dimeric N-terminal fragment. Upon reconstitution of labeled L7/L12 with ribosomal cores, however, the motion associated with the dimeric N-terminal domain is greatly diminished while the facile motion of the C-terminal domains is almost unchanged.

Dimer/monomer equilibrium and domain separations of Escherichia coli ribosomal protein L7/L12.

The dimer to monomer equilibrium and interdomain separations of cysteine variants of L7/L12 have been investigated using fluorescence spectroscopy. Steady-state polarization measurements on cysteine containing variants of L7/L12, labeled with 5-(iodoacetamido)fluorescein, demonstrated dimer to monomer dissociation constants near 30 nM for variants labeled at position 33, in the N-terminal domain, and positions 63 and 89, in the C-terminal domain. A dissociation constant near 300 nM was determined for a variant labeled at position 12, in the N-terminal domain. The polarization of a labeled C-terminal fragment did not change over the range of 200 microM to 1 nM, indicating that this construct remains monomeric at these concentrations, whereas a dimer to monomer dissociation constant near 300 nM was observed for an FITC labeled N-terminal fragment. Intersubunit fluorescence resonance energy self-transfer was observed when appropriate probes were attached to cysteines at residues 12 or 33, located in the N-terminal domain. Probes attached to cysteines at positions 63 or 89 in the C-terminal domain, however, did not exhibit intersubunit self-transfer. These results indicate that these residues in the C-terminal domains are, on average, separated by greater than 85 A. Intersubunit self-transfer does occur in a C-89 double mutation variant lacking 11 residues in the putative hinge region, indicating that the loss of the hinge region brings the two C-terminal domains closer together. Rapid subunit exchange between unlabeled wild-type L7/L12 and L7/L12 variants labeled in the N-terminal domain was also demonstrated by the loss of self-transfer upon mixing of the two proteins.

Tetramethylrhodamine dimer formation as a spectroscopic probe of the conformation of Escherichia coli ribosomal protein L7/L12 dimers.

The fluorescent probe tetramethylrhodamine iodoacetamide was attached to cysteine residues substituted at various specific locations in full-length and deletion variants of the homodimeric Escherichia coli ribosomal protein L7/L12. Ground-state tetramethylrhodamine dimers form between the two subunits of L7/L12 depending upon the location of the probe. The formation of tetramethylrhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the different protein variants. Intersubunit tetramethylrhodamine dimers form when tetramethylrhodamine acetamide is attached to two different sites in the N-terminal domain of the L7/L12 dimer (residues 12 or 33), but not when attached to sites in the C-terminal domain (residues 63, 89, or 99). The tetramethylrhodamine dimers do form at sites in the C-terminal domain in L7/L12 variants that contain deletions of 11 or 18 residues within the putative flexible hinge that separates the N- and C-terminal domains. The tetramethylrhodamine dimers disappear rapidly (within 5 s) upon addition of excess unlabeled wild-type L7/L12. It appears that singly labeled L7/L12 dimers are formed by exchange with wild-type dimers. Binding of L7/L12:tetramethylrhodamine cysteine 33 or cysteine 12 dimers either to L7/L12-depleted ribosomal core particles, or to ribosomal protein L10 alone, results in disappearance of the 518-nm absorption band. This result implies a conformational change in the N-terminal domain of L7/L12 upon its binding to the ribosome, or to L10.

Location and domain structure of Escherichia coli ribosomal protein L7/L12: site specific cysteine crosslinking and attachment of fluorescent probes.

Five different variants of L7/L12 containing single cysteine substitutions, two in the N-terminal (NTD) and three in the C-terminal domain (CTD), were produced, modified with [125I]N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio) propionamide ([125I]APDP), a sulfhydryl-specific, heterobifunctional, cleavable photo-cross-linking reagent, and reconstituted into ribosomes. These were irradiated, the total proteins were extracted and reductively cleaved, and the cross-linked proteins were identified. The effect of zero-length disulfide cross-linking on binding and activity was also determined. The same sites in L7/L12 were used to attach a rhodamine dye. The formation of ground-state rhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the free protein and in complexes with L10. The three sites in the CTD, but not the N-terminal sites, cross-linked to L2 and L5 and to 30S proteins S2, S3, S7, S14, and S18 in a manner influenced by elongation factors. Binding to the ribosome and, therefore, function were blocked by zero-length cross-linking within the NTD, but not the CTD. Binding also disrupted rhodamine dimers in the NTD. No rhodamine dimers formed in the CTD.

Different molecular forms of rat kidney gp330, the dominant autoantigen of active Heymann nephritis.

The primary structure, consisting of 1650 amino acid residues, of the C-terminal end of the dominant autoantigen of active Heymann Nephritis, gp330, from rat kidney was obtained by cloning and sequencing of cDNA clones. Comparison of this sequence with the previously published sequences of fragments of the C-terminal end of gp330 [Raychowdhury, Niles, McCluskey and Smith (1989) Science 244, 1163-1165] revealed certain differences in their primary structures. These differences included several single amino acid substitutions, replacement of a stretch of 15 amino acid residues by a different stretch of six amino acid residues, and different lengths of cytoplasmic domain (188 versus 213 amino acid residues). These findings of two different primary structures of gp330 provide direct evidence for the existence of two molecular forms of gp330.

Escherichia coli ribosomal protein L7/L12 dimers remain fully active after interchain crosslinking of the C-terminal domains in two orientations.

Cysteine site-directed mutagenesis was used to create variants of Escherichia coli ribosomal protein L7/L12 that have single cysteine substitutions, at residues 63 or 89, located in different exposed loops in the structure of the globular C-terminal domain indicated by the crystallographic structure. That structure shows a possible dimer interaction in which the two sites of cysteine substitution appear to be too distant for disulfide bond formation. After mild oxidation in solution both of the overexpressed purified cysteine-substituted proteins formed interchain disulfide crosslinked dimers in high yield. Both crosslinked dimers were fully active in restoring activity in poly(U)-directed polyphenylalanine synthesis to ribosomal core particles depleted of wild-type L7/L12. These results show that the two C-terminal domains have independent mobility. The activity of dimeric L7/L12 does not require the independent movement of the two globular C-terminal domains in an L7/L12 dimer; moreover, it appears independent of their mutual orientation when joined by crosslinking at the two loops. A third variant with a cysteine substitution at residue 33 near the junction between the alpha-helical N-terminal domain and the flexible hinge was prepared and tested. This protein was active in the protein synthesis assay in the reduced state. Oxidation produced the interchain crosslinked dimer in high yield, but this crosslinked dimer was inactive in polyphenylalanine synthesis. The inactivation was due to the inability of the Cys33-Cys33 oxidized dimer to bind to the core particle.

Primary structure of the cDNA 5'-terminal region encoding the N-terminal domain of the rabbit muscle alpha-actinin subunit.

A number of cDNA clones have been obtained in summary encoding the N-terminal domain containing 286 amino acid residues of the rabbit skeletal muscle alpha-actinin subunit. Alpha-Actinin cDNA clones were isolated from specific cDNA libraries using the primer extension method for synthesis of the first cDNA chain. A strong stop signal for AMV reverse transcriptase in the 5'-terminal region of mRNA of alpha-actinin was found. It seems there is a G+C rich region (93% G+C nucleotides) including a continuous sequence of 23 G and C nucleotides encoding 6 glycine residues.

Primary structure of rat liver elongation factor 2 deduced from the cDNA sequence.

A number of cDNA clones in summary encoding 700 amino acid residues from the N-end of rat liver elongation factor 2 (EF-2) and including 49 nucleotides of the 5'-untranslated mRNA region have been obtained. EF-2 cDNA clones were isolated from gradually constructed small (1000-5000 clones) specific cDNA libraries using the primer extension method for synthesis of the first cDNA chain. The complete primary structure of cDNA and protein EF-2 from rat liver was derived taking into account the primary structure of the 3'-terminal region EF-2 cDNA previously reported [(1986) Proc. Natl. Acad. Sci. USA 83, 4978-4982]. Comparison of this cDNA with hamster cDNA has shown that (i) the base sequences had a 89.7% homology while that of the 5'-untranslated region was 73%; (ii) there are two amino acid replacement in rat liver EF-2 as compared with hamster EF-2.