Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins.

BACKGROUND: DNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators. RESULTS: This study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1ß and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as "threads" by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions. CONCLUSION: KfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.

Alpha-helix nucleation by a calcium-binding peptide loop.

A 12-residue peptide AcDKDGDGYISAAENH2 analogous to the third calcium-binding loop of calmodulin strongly coordinates lanthanide ions (K = 10(5) M-1). When metal saturated, the peptide adopts a very rigid structure, the same as in the native protein, with three last residues AAE fixed in the alpha-helical conformation. Therefore, the peptide provides an ideal helix nucleation site for peptide segments attached to its C terminus. NMR and CD investigations of peptide AcDKDGDGYISAAEAAAQNH2 presented in this paper show that residues A13-Q16 form an alpha-helix of very high stability when the La3+ ion is bound to the D1-E12 loop. In fact, the lowest estimates of the helix content in this segment give values of at least 80% at 1 degreesC and 70% at 25 degreesC. This finding is not compatible with existing helix-coil transition theories and helix propagation parameters, s, reported in the literature. We conclude, therefore, that the initial steps of helix propagation are characterized by much larger s values, whereas helix nucleation is even more unfavorable than is believed. In light of our findings, thermodynamics of the nascent alpha-helices is discussed. The problem of CD spectra of very short alpha-helices is also addressed.

A comparative CD and fluorescence study of a series of model calcium-binding peptides.

Lanthanide-saturated peptides analogous to calcium-binding loops of EF-hand proteins can be used to stabilize the alpha-helical structure of peptide or protein segments attached to their C-termini. To study conformational properties of such loop-containing hybrids it is necessary to produce them in bacteria. In peptides obtained in this way the helix will be destabilized by the negatively charged C-terminal alpha-carboxyl groups. We propose to block them by the homoserine lactone. The results presented in this paper indicate that the presence of the lactone even at the C-terminus of the loop does not have any negative effect on the loop helix-nucleation ability. On the other hand, the presence of the alpha-NH3+ at the loop N-terminus leads to a drop of metal-binding constant and loss of the rigid structure of the alpha-helical segment of the loop. The alpha-amino group separated by one glycine residue from the loop N-terminus should also be avoided because it perturbs the conformation of the N-terminal part of the loop and may reduce the loop affinity to lanthanide ions.

Molecular cloning and expression in Escherichia coli of a gene coding for bovine S100A1 protein and its Glu32-->Gln and Glu73-->Gln mutants.

Calcium binding S100A1 protein consists of two S100 alpha subunits. On the basis of sequence homology to other S100 proteins it is believed that the binding loops are formed by amino-acid residues 19-32 and 62-73 of S100 alpha polypeptide chain. In the oxidized form of the protein the subunits are linked covalently with each other by a disulphide bond between their Cys85 residues. A synthetic gene coding for bovine S100 alpha subunit was constructed and cloned into a derivative of pAED4 plasmid. The gene was expressed in Escherichia coli utilizing the T7 expression system. The expression products were purified and identified using mass spectrometry and by sequencing of their N- and C-termini. Three different forms (a, b, and c) of S100 alpha were produced: with the native sequence, with the initiator methionine at the N-terminus, and with an additional alanine at the C-terminus as well as with the initiator methionine. The material was partly oxidized. Interestingly, only the homodimers of a, b, and c species were formed. The total yield of the protein was about 50 mg/l of culture. Genes coding for Glu32-->Gln and Glu73-->Gln mutants of S100 alpha were obtained by site-directed mutagenesis and expressed in the same system. In both cases similar mixtures of oxidized and reduced a, b, and c species have been obtained. The total yield of E73Q mutant is similar to that of the native protein and that of E32Q lower by about a half. As expected, the mutants of S100 alpha subunits bind only one calcium ion.