Crystal state conformation of three model monomer units for the beta-bend ribbon structure

The molecular and crystal structures of three compounds, representing the repeating units of the -bend ribbon (an approximate 310-helix, with an intramolecular hydrogen-bonding donor every two residues), have been determined by x-ray diffraction. They are Boc-Aib-Hib-NHBzl, Z-Aib-Hib-NHBzl, and Z-L-Hyp-Aib-NHMe (Aib, -aminoisobutyric acid; Bzl, benzyl; Boc, t-butyloxycarbonyl; Hyp, hydroxyproline Hib, -hydroxyisobutyric acid; Z, benzyloxycarbonyl). The two former compounds are folded in a -bend conformation: type III (III) for Boc-Aib-Hib-NHBzl, while type II (II) for the Z analogue. Conversely, the structure of Z-L-Hyp-Aib-NHMe, although not far from a type II -bend, is partially open.

Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu

A sensitive dimerization assay for DNA binding proteins has been developed using gene fusion technology. For this purpose, we have engineered a gene fusion using protein A gene of Staphylococcus aureus and C gene, the late gene transactivator of bacteriophage Mu. The C gene was fused to the 3' end of the gene for protein A to generate an A- C fusion. The overexpressed fusion protein was purified in a single step using immunoglobulin affinity chromatography. Purified fusion protein exhibits DNA binding activity as demonstrated by electrophoretic mobility shift assays. When the fusion protein A-C was mixed with C and analyzed for DNA binding, in addition to C and A-C specific complexes, a single intermediate complex comprising of a heterodimer of C and A-C fusion proteins was observed. Further, the protein A moiety in the fusion protein A-C does not contribute to DNA binding as demonstrated by proteolytic cleavage and circular dichroism (CD) analysis. The assay has also been applied to analyze the DNA binding domain of C protein by generating fusions between protein A and N- and C-terminal deletion mutants of C. The results indicate a role for the region towards the carboxy terminal of the protein in DNA binding. The general applicability of this method is discussed.