The X-ray structure of Escherichia coli TEM1 beta-lactamase has been refined to a crystallographic R-factor of 16.4% for 22,510 reflections between 5.0 and 1.8 A resolution; 199 water molecules and 1 sulphate ion were included in refinement. Except for the tips of a few solvent-exposed side chains, all protein atoms have clear electron density and refined to an average atomic temperature factor of 11 A2. The estimated coordinates error is 0.17 A. The substrate binding site is located at the interface of the two domains of the protein and contains 4 water molecules and the sulphate anion. One of these solvent molecules is found at hydrogen bond distance from S70 and E166. S70 and S130 are hydrogen bonded to K73 and K234, respectively. It was found that the E. coli TEM1 and Staphylococcus aureus PC1 beta-lactamases crystal structures differ in the relative orientations of the two domains composing the enzymes, which result in a narrowed substrate binding cavity in the TEM1 enzyme. Local but significant differences in the vicinity of this site may explain the occurrence of TEM1 natural mutants with extended substrate specificities.

The antibiotic bleomycin, a strong DNA cutting agent, is naturally produced by actinomycetes which have developed a resistance mechanism against such a lethal compound. The crystal structure, at 2.3 A resolution, of a bleomycin resistance protein of 14 kDa reveals a structure in two halves with the same alpha/beta fold despite no sequence similarity. The crystal packing shows compact dimers with a hydrophobic interface and involved in mutual chain exchange. Two independent solution studies (analytical centrifugation and light scattering) showed that this dimeric form is not a packing artefact but is indeed the functional one. Furthermore, light scattering also showed that one dimer binds two antibiotic molecules as expected. A crevice located at the dimer interface, as well as the results of a site‐directed mutagenesis study, led to a model wherein two bleomycin molecules are completely sequestered by one dimer. This provides a novel insight into antibiotic resistance due to drug sequestering, and probably also into drug transport and excretion.

Hinge prolines can be considered as 'quaternary structure helpers'. The presence of a proline should be considered when searching for a determinant of oligomerization with arm exchange and could be used to engineer synthetic oligomers or to displace a monomers to oligomers equilibrium by mutation of this proline residue.

We have developed an efficient method that electrically introduces DNA into intact yeast cells. Saccharomyces cerevisiae was used as a model in order to optimize the transformation protocol. Transformation efficiencies of 10(7) transformants/micrograms of plasmid DNA were obtained with a square wave electric pulse of 2.7 kV/cm during 15 milliseconds. The technique is simple and rapid. Even small quantities of DNA (100 pg) can be used to transform 10(8) cells. Important parameters are the pulse field strength and duration. Pretreatment of the yeast cells in the early phase of exponential growth with dithiothreitol increases transformation efficiency. The method has been successfully applied to various strains of S. cerevisiae as well as to other types of yeast.