Hans-Werner Adolph

MultiLoc: prediction of protein subcellular localization using N-terminal targeting sequences, sequence motifs and amino acid composition

MOTIVATIONnFunctional annotation of unknown proteins is a major goal in proteomics. A key annotation is the prediction of a proteins subcellular localization. Numerous prediction techniques have been developed, typically focusing on a single underlying biological aspect or predicting a subset of all possible localizations. An important step is taken towards emulating the protein sorting process by capturing and bringing together biologically relevant information, and addressing the clear need to improve prediction accuracy and localization coverage.nnnRESULTSnHere we present a novel SVM-based approach for predicting subcellular localization, which integrates N-terminal targeting sequences, amino acid composition and protein sequence motifs. We show how this approach improves the prediction based on N-terminal targeting sequences, by comparing our method TargetLoc against existing methods. Furthermore, MultiLoc performs considerably better than comparable methods predicting all major eukaryotic subcellular localizations, and shows better or comparable results to methods that are specialized on fewer localizations or for one organism.nnnAVAILABILITYnhttp://www-bs.informatik.uni-tuebingen.de/Services/MultiLoc/

Mono- and binuclear Zn-β-lactamase from Bacteroides fragilis: catalytic and structural roles of the zinc ions

The Bacteroides fragilis Zn‐β‐lactamase is active with a mono‐ and a binuclear zinc site. The apoenzyme produced by removal of both Zn ions does not recover full activity upon readdition of Zn2+ in contrast to an active mono‐Zn form prepared at pH 6.0. Differences in k cat values observed are substrate‐dependent implying distinct mechanisms for the mono‐ and binuclear species. The substrate profile of a Zn,Cd hybrid obtained by selective exchange of one zinc ion is different from that of the Zn2 enzyme with a remarkable 15‐fold increased activity with cefoxitin as substrate.

Studies on ternary metallo-β lactamase-inhibitor complexes using electrospray ionization mass spectrometry

Metallo-β-lactamases (MBLs) are targets for medicinal chemistry as they mediate bacterial resistance to β-lactam antibiotics. Electrospray-ionization mass spectrometry (ESI-MS) was used to study the inhibition by a set of mercaptocarboxylates of two representative MBLs with different optimal metal stoichiometries for catalysis. BcII is a dizinc MBL (Class B1), whilst the CphA MBL (Class B2) exhibits highest activity with a single zinc ion in the active site. Experimental parameters for the detection of the metallo-enzyme and the metallo-enzyme-inhibitor complexes were evaluated and optimized. Following investigations on the stoichiometry of metal binding, the affinity of the inhibitors was investigated by measuring the relative abundance of the complex compared to the metalloprotein. The results for the BcII enzyme were in general agreement with solution assays and demonstrated that the inhibitors bind to the dizinc form of the BcII enzyme. The results for the CphA(ZnII) complex unexpectedly revealed an increased affinity for the binding of a second metal ion in the presence of thiomandelic acid. The results demonstrate that direct ESI-MS analysis of enzyme:inhibitor complexes is a viable method for screening inhibitors and for the rapid assay of the enzyme:metal:inhibitor ratios.