Biomedical subjects

S G Eagle

Publications and source records attributed to S G Eagle.

4 recordsLinked to original sources

PubMed1994

Structural similarity of a developmentally regulated bacterial spore coat protein to beta gamma-crystallins of the vertebrate eye lens.

The solution structure of Ca(2+)-loaded protein S (M(r) 18,792) from the Gram-negative soil bacterium Myxococcus xanthus has been determined by multidimensional heteronuclear NMR spectroscopy. Protein S consists of four internally homologous motifs, arranged to produce two domains with a pseudo-twofold symmetry axis, overall resembling a triangular prism. Each domain consists of two topologically inequivalent "Greek keys": the second and fourth motifs form standard Greek keys, whereas the first and third motifs each contain a regular alpha-helix in addition to the usual four beta-strands. The structure of protein S is similar to those of the vertebrate eye lens beta gamma-crystallins, which are thought to be evolutionarily related to protein S. Both protein S and the beta gamma-crystallins function by forming stable multimolecular assemblies. However, protein S possesses distinctive motif organization and domain packing, indicating a different mode of oligomerization and a divergent evolutionary pathway from the beta gamma-crystallins.

Animals↗

PubMed1994

Unusual helix-containing greek keys in development-specific Ca(2+)-binding protein S. 1H, 15N, and 13C assignments and secondary structure determined with the use of multidimensional double and triple resonance heteronuclear NMR spectroscopy.

Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone and side-chain 1H, 15N, and 13C resonance assignments of calcium loaded Myxococcus xanthus protein S (173 residues). Of the range of constant-time triple resonance experiments recorded, HNCACB and CBCA(CO)NH, which correlate C alpha and C beta with backbone amide resonances of the same and the succeeding residue respectively, proved particularly useful in resolving assignment ambiguities created by the 4-fold internal homology of the protein S amino acid sequence. Extensive side-chain 1H and 13C assignments have been obtained by analysis of HCCH-TOCSY and 15N-edited TOCSY-HMQC spectra. A combination of NOE, backbone amide proton exchange, 3JNH alpha coupling constant, and chemical shift data has been used to show that each of the protein S repeat units consists of four beta-strands in a Greek key arrangement. Two of the Greek keys contain a regular alpha-helix between the third and fourth strands, resulting in an unusual and possibly unique variation on this common folding motif. Despite similarity between two nine-residue stretches in the first and third domains of protein S and one of the Ca(2+)-binding sequences in bovine brain calmodulin [Inouye, S., Franceschini, T., & Inouye, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6829-6833], the protein S topology in these regions is incompatible with an EF-hand calmodulin-type Ca(2+)-binding site.

Amino Acid Sequence↗

PubMed1994

NMR-derived three-dimensional solution structure of protein S complexed with calcium.

BACKGROUND: Protein S is a developmentally-regulated Ca(2+)-binding protein of the soil bacterium Myxococcus xanthus. It functions by forming protective, multilayer spore surface assemblies which may additionally act as a cell-cell adhesive. Protein S is evolutionarily related to vertebrate lens beta gamma-crystallins. RESULTS: The three-dimensional solution structure of Ca(2+)-loaded protein S has been determined using multi-dimensional heteronuclear NMR spectroscopy. (Sixty structures were calculated, from which thirty were selected with a root mean square difference from the mean of 0.38 A for backbone atoms and 1.22 A for all non-hydrogen atoms.) The structure was analyzed and compared in detail with X-ray crystallographic structures of beta gamma-crystallins. The two internally homologous domains of protein S were compared, and hydrophobic cores, domain interfaces, surface ion pairing, amino-aromatic interactions and potential modes of multimerization are discussed. CONCLUSIONS: Structural features of protein S described here help to explain its overall thermostability, as well as the higher stability and Ca2+ affinity of the amino-terminal domain relative to the carboxy-terminal domain. Two potential modes of multimerization are proposed involving cross-linking of protein S molecules through surface Ca(2+)-binding sites and formation of the intramolecular protein S or gamma B-crystallin interdomain interface in an intermolecular content. This structural analysis may also have implications for Ca(2+)-dependent cell-cell interactions mediated by the vertebrate cadherins and Dictyostelium discoideum protein gp24.

Amino Acid Sequence↗

PubMed1992

Cell-free synthesis of the branched RNA-linked msDNA from retron-Ec67 of Escherichia coli.

msDNA-Ec67 is produced in a clinical strain of Escherichia coli and composed of a 67-base single-stranded DNA, which is linked to the 2'-OH group of the 15th rG residue of a 58-base RNA molecule by a 2',5'-phosphodiester linkage (Lampson, B. C., Sun, J., Hsu, M.-Y., Vallejo-Ramirez, J., Inouye, S., and Inouye, M. (1989) Science 243, 1033-1038). The production of msDNA-Ec67 is dependent upon retron-Ec67, which consists of the msr-msd region and the gene for reverse transcriptase (RT). These two elements were separately cloned into plasmids; p67-BHO.6 contained the msr-msd region and pRT-67 contained the RT gene under the lpp-lac promoter-operator. msDNA-Ec67 was produced only when cells were transformed with both plasmids. In addition, msDNA-Ec67 was synthesized in a cell-free system using total RNA prepared from cells harboring plasmid p67-BHO.6 and purified Ec67-RT. Using this cell-free system, the priming reaction, during initiation of DNA synthesis, was demonstrated to be a specific template-directed event; only dTTP was incorporated into a 132-base precursor RNA yielding a 133-base compound. This specific dT addition could be altered to dA or dC by simply substituting the 118th A residue of the putative msr-msd transcript with a T or G residue. The priming reaction was blocked when A was substituted for G at the 15th residue of the precursor RNA transcript, which corresponds to the branched rG residue in msDNA. DNA chain elongation could be terminated by adding ddNTP in the cell-free system, forming a sequence ladder. The DNA sequence determined from this ladder completely agreed with the msDNA sequence. The RT extension reaction was completely blocked when the RNA preparation was treated with RNase A but not when the preparation was treated with DNase. This clearly demonstrates that RNA but not DNA is responsible for the msDNA production. A part of the fully extended cell-free product contained a 13-base RNA strand resistant to RNase A, which is consistent with the previously proposed model. In this model, the 5'-end sequence of the msr-msd transcript (a2; bases 1-13) forms a duplex with the 3'-end sequence (a1) of the same transcript, thus serving as a primer, as well as a template for msDNA synthesis by RT. Our results are inconsistent with a model recently proposed by Lease and Yee (Lease, R. A., and Yee, T. (1991) J. Biol. Chem. 266, 14497-14503).

Base Sequence↗