Purification of F plasmid-encoded native TraC from Escherichia coli by affinity chromatography on calmodulin Sepharose.

We have enriched several native bacterial proteins from Escherichia coli by chromatography on the immobilized eukaryotic Ca(2+)-binding protein, calmodulin. These bacterial proteins bound in a Ca(2+)-dependent manner to calmodulin, and were released by the addition of the Ca(2+)-chelator, EGTA, similar to many eukaryotic calmodulin-binding proteins. One of the bacterial proteins, F factor-encoded TraC, was purified to apparent homogeneity by an additional chromatographic step, anion exchange chromatography on MonoQ.

Structure/function analysis of spinalin, a spine protein of Hydra nematocysts.

The nematocyst capsules of the cnidarians are specialized explosive organelles that withstand high osmotic pressures of approximately 15 MPa (150 bar). A tight disulfide network involving cysteine-rich capsule wall proteins, like minicollagens and nematocyst outer wall antigen, characterizes their molecular composition. Nematocyst discharge leads to the expulsion of a long inverted tubule that was coiled inside the capsule matrix before activation.

Functional studies on recombinant domains of Mac-2-binding protein.

Mac-2-binding protein (M2BP) is a secreted glycoprotein suggested to have a role in host defense. It forms linear and ring-shaped oligomers, with each ring segment being composed of two monomers. We have produced recombinant human M2BP fragments comprising domains 1 and 2 (M2BP-1,2) and domains 3 and 4 (M2BP-3,4) in 293 human kidney cells to characterize structural and functional properties of M2BP.

Sarcolipin, the shorter homologue of phospholamban, forms oligomeric structures in detergent micelles and in liposomes.

The human 31-amino acid integral membrane protein sarcolipin (SLN), which regulates the sarcoplasmic reticulum Ca-ATPase in fast-twitch skeletal muscle, was chemically synthesized. Appropriate synthesis and purification strategies were used to achieve high purity and satisfactory yields of this hydrophobic and poorly soluble protein. Structural and functional properties of SLN were analyzed and compared with the homologous region of human phospholamban (PLB) comprising residues Ala(24)-Leu(52) (PLB-(24-52)), the regulatory protein of the cardiac sarcoplasmic reticulum Ca-ATPase.

Toward a high-resolution structure of phospholamban: design of soluble transmembrane domain mutants.

Determination of a high-resolution structure of the phospholamban (PLB) transmembrane domain by X-ray crystallography or NMR is handicapped by the hydrophobic nature of the peptide. Interestingly, the crystal structure of the five-stranded parallel coiled-coil oligomerization domain from cartilage oligomeric matrix protein (COMPcc) shows marked similarities to a model proposed for the pentameric transmembrane domain of PLB. Contrary to the putative coiled-coil domain of PLB, COMPcc contains mostly hydrophilic amino acids on the surface, resulting in a soluble molecule.

Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide. Evidence for a dithiol-disulfide equilibrium and implications for redox regulation.

Calcineurin (CaN) is a Ca2+-and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe-Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated.

Overexpression, purification and characterization of Dictyostelium calcineurin A.

The catalytic subunit of Ca2+/calmodulin-dependent protein phosphatase (calcineurin A) was overexpressed about 50-fold in Dictyostelium discoideum cells transformed with a vector containing the cDNA for D. discoideum calcineurin A under control of the actin-6 promoter. In crude lysates from the overexpressing cell line, high Ca2+/calmodulin-stimulated phosphatase activity was detected.

Primary structure, expression and developmental regulation of a Dictyostelium calcineurin A homologue.

cDNA clones for the catalytic subunit of Ca2+/calmodulin(CaM)-dependent protein phosphatase (calcineurin A, protein phosphatase 2B) from Dictyostelium discoideum were isolated by functional screening of a lambda gt11 lysogen expression library with labeled Dictyostelium CaM. A complete cDNA of 2146 bp predicts a protein of 623 amino acids with homology to calcineurin A from other organisms and a similar molecular architecture. However, the Dictyostelium protein contains N-terminal and C-terminal extra domains causing a significantly higher molecular mass than found in any of its known counterparts.