No description available.

As shown in previous studies Streptomyces lividans enzymatically inactivates fusidic acid by specific esterase FusH giving rise to the 16ß-OH derivative, which sponaneously converts to the lactone.in this work it was shown that S. lividans further modifies fusidic acid and both derivatives, which resulted in several new related substances. The two intermediates (La, Lb) were isolated from the culture filtrate of S. lividans, which was grown in the presence of fusidic acid. The differences in their chemical structures indicate the involvement of multiple enzyme reactions related to hydroxylation, hydration, dehydrogenation and isomerization. Several enzymes were identified and two of them (FusG, FusB) were partially characterized. According to their characteristics and the structures of isolated intermediates, the identified enzymes which are involved in biotranformation are conceivably related to the ones implicated in ß oxidation. The biotransformation of fusidic acid and its derivatives by S. lividans is so far unique, since characterized substances La and Lb have not been found in either fusidic acid-producing or fusidic acid-resistant microorganisms.

No description available.

No description available.

The KdpFABC complex from E. coli functions as a high affinity K uptake system and belongs to the superfamily of P-type ATPases. So far, no information is available about the orientation of the subunits within the complex as well as its oligomeric state. By chemical crosslinking, gel filtration, electron transmission microscopy and single particle FRET analysis this study shows that the KdpFABC complex occurs as a homodimer with a dissociation constant between 30 to 50 nM. Furthermore, by means of single particle analysis of transmission electron micrographs, the solution structure of the complex at 1.9 nm resolution could be solved, thus providing the first structural analysis resolving all subunits of the holoenzyme. Based on crystal structures, it is generally assumed that P-type ATPases undergo large domain movements during catalysis. However, these conformational changes have never been shown directly. By use of single molecule FRET with alternating laser excitation, distance changes could be measured directly within KdpB during ATP hydrolysis. With this technique, distances and dwell times were determined for three conformational states in the working enzyme as well as in the orthovanadate- and the OCS-inhibited state.

1. Aufgrund der Resultate mit der Mutante MM10, die trotz Disulfidbrücke noch unverminderte Aktivität und Rotation zeigte, wurde der Frage nachgegangen, ob der EF1-Komplex in der Lage ist, die Rotation durch einen Bruch der alpha-helikalen Struktur von gamma oder durch Rotation um eine Einfachbindung der Disulfidbrücke aufrechtzuerhalten. Quervernetzungen vom Hexagon mit gamma in der Mitte und am unteren Ende konnten das Enzym blockieren. Von den vier betrachteten Mutanten KG11, MM26, MM25 und MM24 fiel der MM26 bereits nach der Isolierung raus. Der MM25 wies nicht mehr als 70% Quervernetzung auf. Bei dem KG11 und dem MM24 konnte jedoch eine 99%-ige Quervernetzung nachgewiesen werden. Mit diesen zwei Cystein-Doppelmutanten wurden weitere Quervernetzungen gefunden, die ebenso wie der MM10 aktiv nach Oxidation sind, aber tiefer im Enzym liegen und die ATP-Hydrolyse trotz Blockade durch Quervernetzung aufrecht erhalten. Es konnte gezeigt werden, dass eine Rotation um die Einzelbindungen innerhalb der Disulfidbrücke unwahrscheinlich ist, und daher die Aktivität des quervernetzten Enzyms nur durch eine Aufwindung der gamma-Helix erklärt werden kann. 2. Die Voraussetzung für ein EFOF1-Kostrukt zum optischen Nachweis der Relativrotation unter Synthesebedingungen war der Einbau von zwei verschiedenen Tags zur spezifischen Bindung. Von den vier Mutanten SE3, SE4, SW7 und WH1 zeigten die beiden SE-Mutanten keine Stabilität bei der Isolierung im Bezug auf die Kopplung zwischen FO- und F1-Teil. Mit dem SW7-EFOF1 wurde eine Mutante gefunden, die mit einer guten Aktivitäts- und Rotationsausbeute nach einer Aufreinigung mittels Streptactin-Affinitätchromatographie durch ihre Stabilität als Ausgangspunkt für das Rotationsexperiment unter ATP-Synthese dienen kann. Der WH1, dessen atp-Operon dem des SW7 gleicht, brachte trotz seines veränderten Vektorursprungs keine Verbesserung.

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No description available.

No description available.

Voltage gated potassium channels (Kv) play a key role in the nervous system- not only due to their involvement in the action potential. Vertebrates express four subtypes, which are termed Kv1, Kv2, Kv3 and Kv4, respectively. Tsha3 is a Kv1 channel which was originally isolated from brain tissue of rainbow trout (Oncorhynchus mykiss). This channel possesses an unique amino terminus and a characteristic amino acid sequence in the T1 domain, which is engaged in the oligomerization of Kv α-subunits and is thus involved into the segregation of subfamilies. The two major goals of this thesis were the structural and functional characterization of the N-terminal cytosolic domain of Tsha3 as well as the invention of a system to gain data about the functional dynamics of full length Kv channels. Molecular biological techniques were used to isolate mRNA from trout brains, to transcribe it into cDNA and clone it into vectors. DNA from such plasmids was ligated into expression vectors for heterologous expression in E. coli, P. pastoris and Sf21 cells, with concomitant fusion of marker proteins (GFP or DsRed) or tags (6 x HisTag or StrepTagII) due to the individual experiment. Protein was overexpressed in E. coli and affinity purified to analyze separated domains with biochemical (SDS-PAGE and Western Blot, Pull-Down-Assay or Dot-Blot-Assay) or biophysical (CD-spectroscopy, EPR spectroscopy) efforts. The P. pastoris system to express Tsha1 was established, to generate a system for future EPR-measurements of whole Kv channels. Heterologous expression of Kv1α (Tsha3 and Tsha1) and the core domain of Kvβ in Sf21 cells was performed to analyze the subcellular distribution of the respective subunits via fluorescence microscope and via subcellular fractionation of cell lysates with downstream biochemical analyses (SDS-PAGE and Western Blot). Furthermore the gating of diverse fusion constructs of Tsha3 in co-expressions and the gating of diverse cystein substitution mutants of Tsha1 were measured via path-clamp recordings in whole cell modus. The structural analyses of the N-terminal cytosolic domain (NCD) of Tsha3 revealed that the 128 amino acid containing part before the T1-domain (Tsha3-NT) can be structurally divided into three parts of different structure and mobility. The most outward part possesses a very high mobility and is putatively unfolded as random coil. This section is expected to express no tertiary contacts. The middle part of Tsha3-NT is structured in α-helices and β-sheets and thus slightly immobile. This folded part is also assumed to build no tertiary structure and to be exposed into the cytosol. The third, which is directly neighboring the T1 domain, has the most restricted mobility of Tsha3-NT. It consists predominantly of α-helices and exhibits a tertiary structure, putatively with the T1 domain. Tsha3-NCD self-tetramerizes and oligomerizes with Tsha1, although mutations exist in Tsha3 in conserved amino acids, which were reported to function in subfamily specific hetero-tetramerization. Thus it is proven, that Tsha3 takes part in the segregation into the Kv1 subfamily. Furthermore, Tsha3 interacts with the core domain of Kvβ2 although there are also mutations in the reported consensus sequence for interaction. Association of Kvβ2 in co-expression studies directs Tsha3-DsRed fusion constructs from internal vesicular structures into the cell membrane. But the fusion with DsRed is leading to a loss of function of Tsha3 which cannot be rescued by co-expression of the chaperone Kvβ2. But- without fusion of marker proteins- Tsha3 was identified as an outward rectifier in a cooperative Bachelor Thesis. These structural data lead to the assumption, that Tsha3-NT exhibits lateral interactions and especially the helical but mobile middle part of the N-terminus can play such a role. Due to the localization next to the membrane, interactions with membrane proteins- putatively with protein cascades are possible. Although Tsha3-NT contains no reported interaction domains for protein-protein interactions, follow-up experiments should be performed to shed light on this interesting question. Tsha1 C30S C31S C180S C224A C239S C389S C424S C476S is a complete cysteine free mutant, which was identified as a functional voltage-gated potassium channel. It was expressed in and purified from eukaryotic cells (P. pastoris) and therefore it can be assumed to be properly folded and modified. After a slight optimization of the features of expression, this system can be used to reconstitute Tsha1 channels into liposomes and use them for Freeze Quench EPR to gain structural information about a Kv1 channel in the open as well as in the closed state. This is the first report of the establishment of a full length Kv for studies of structure and functional dynamics experiments.