Biomedical subjects

Lars Kuerschner

Publications and source records attributed to Lars Kuerschner.

4 recordsLinked to original sources

PubMed2004

Polyene-lipids: a new tool to image lipids.

Microscopy of lipids in living cells is currently hampered by a lack of adequate fluorescent tags. The most frequently used tags, NBD and BODIPY, strongly influence the properties of lipids, yielding analogs with quite different characteristics. Here, we introduce polyene-lipids containing five conjugated double bonds as a new type of lipid tag. Polyene-lipids exhibit a unique structural similarity to natural lipids, which results in minimal effects on the lipid properties. Analyzing membrane phase partitioning, an important biophysical and biological property of lipids, we demonstrated the superiority of polyene-lipids to both NBD- and BODIPY-tagged lipids. Cells readily take up various polyene-lipid precursors and generate the expected end products with no apparent disturbance by the tag. Applying two-photon excitation microscopy, we imaged the distribution of polyene-lipids in living mammalian cells. For the first time, ether lipids, important for the function of the brain, were successfully visualized.

3T3-L1 Cells↗

PubMed2003

Probing the catalytic activity of a cell division-specific transpeptidase in vivo with beta-lactams.

Penicillin-binding protein 3 (PBP3; also called FtsI) is a transpeptidase that catalyzes cross-linking of the peptidoglycan cell wall in the division septum of Escherichia coli. To determine whether the catalytic activity of PBP3 is activated during division, we assayed acylation of PBP3 with three beta-lactams (cephalexin, aztreonam, and piperacillin) in growing cells. Acylation of PBP3 with cephalexin, but not aztreonam or piperacillin, appeared to be stimulated by cell division. Specifically, cephalexin acylated PBP3 about 50% faster in a population of dividing cells than in a population of filamentous cells in which division was inhibited by inactivation or depletion of FtsZ, FtsA, FtsQ, FtsW, or FtsN. However, in a simpler in vitro system using isolated membranes, acylation with cephalexin was not impaired by depletion of FtsW or FtsN. A conflicting previous report that the ftsA3(Ts) allele interferes with acylation of PBP3 was found to be due to the presence of a thermolabile PBP3 in the strain used in that study. The new findings presented here are discussed in light of the hypothesis that the catalytic activity of PBP3 is stimulated by interaction(s) with other division proteins. We suggest that there might be allosteric activation of substrate binding.

Bacterial Proteins↗

PubMed2002

Inhibition of cathepsin G by 2-amino-3,1-benzoxazin-4-ones: kinetic investigations and docking studies.

A series of benzoxazinones was used to investigate the interaction of human cathepsin G with acyl-enzyme inhibitors. With respect to the primary specificity of cathepsin G, inhibitors with hydrophobic or basic residues at position 2 were included in the study. Parameters of the enzyme acylation and deacylation were determined by slow-binding kinetics in the presence of a chromogenic substrate. For selected inhibitors, the time course of the enzyme-catalyzed conversion of the inhibitors was followed. This approach was suitable to elucidate a rate-determining deacylation step. Docking simulations of the noncovalent enzyme-inhibitor complexes were performed and several clusters were analyzed for each inhibitor. The amino acids of the active site that participate in the binding of the inhibitors were determined. The arrangements in several clusters of an inhibitor were not uniform with respect to the orientation by which the inhibitor was bound in the S(1) pocket. Docking of the basic piperazino derivatives 6 and 10 indicated an interaction with Glu 226 at the bottom of the S(1) specificity pocket. The (N-methyl)benzylamino derivative 1 showed the strongest acylation rate (k(on)=1200 M(-1) s(-1)), which was attributed to a high extent of pseudo-productive orientations of the noncovalent preassociation complex.

Benzoxazoles↗

PubMedDate not supplied

Observing structure, function and assembly of single proteins by AFM.

Single molecule experiments provide insight into the individuality of biological macromolecules, their unique function, reaction pathways, trajectories and molecular interactions. The exceptional signal-to-noise ratio of the atomic force microscope allows individual proteins to be imaged under physiologically relevant conditions at a lateral resolution of 0.5-1nm and a vertical resolution of 0.1-0.2nm. Recently, it has become possible to observe single molecule events using this technique. This capability is reviewed on various water-soluble and membrane proteins. Examples of the observation of function, variability, and assembly of single proteins are discussed. Statistical analysis is important to extend conclusions derived from single molecule experiments to protein species. Such approaches allow the classification of protein conformations and movements. Recent developments of probe microscopy techniques allow simultaneous measurement of multiple signals on individual macromolecules, and greatly extend the range of experiments possible for probing biological systems at the molecular level. Biologists exploring molecular mechanisms will benefit from a burgeoning of scanning probe microscopes and of their future combination with molecular biological experiments.

Biophysical Phenomena↗