Scanning and transmission electron microscopy of an unusual soil bacterium: application of the critical point drying method

Bystricky, Vojtech; Fromme, Hans Georg; Pfautsch, M.; Pfefferkorn, G.

doi:10.1016/0047-7206(71)90043-4

Cited by 2 publications

(1 citation statement)

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“…Two other methods are freeze dehydration10 and a critical‐point drying method,11 which are frequently used to prepare samples for electron microscopy. However, these methods are not ideal because it is doubtful whether they completely preserve elemental distributions (although they seem to preserve morphologies relatively well according to electron microscopy images) 16…”

Section: Introductionmentioning

confidence: 99%

Binding Mode of TMC‐95A Analogues to Eukaryotic 20S Proteasome

et al. 2004

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The complex thermodynamics that govern noncovalent protein-ligand interactions are still not fully understood, despite the exponential increase in experimental structural data available from X-ray crystallography and NMR spectroscopy. The eukaryotic 20S proteasome offers an ideal system for such studies as it contains in duplicate three proteolytically active sites with different substrate specificities. The natural product TMC-95A inhibits these proteolytic centers noncovalently with distinct affinities. X-ray crystallographic analysis of the complexes of the yeast proteasome core particle with this natural inhibitor and two synthetic analogues clearly revealed highly homologous hydrogen-bonding networks involving mainly the peptide backbone despite the strongly differentiated binding affinities to the three active sites of the 20S proteasome. The natural product and the two analogues are constrained in a rigid beta-type extended conformation by the endocyclic biaryl clamp, which preorganizes the peptide backbone for optimal adaptation of the ligands to the active site clefts and thus favors the binding processes entropically. However, the biaryl clamp also dictates the orientation of the P1 and P3 residues and their mode of interaction with the protein binding subsites. This limitation is optimally solved in TMC-95A with the conformationally restricted (Z)-prop-1-enyl group acting as P1 residue, at least for the chymotrypsin-like active site; however, it critically affects the inhibitory potencies of the analogues, thus suggesting the use of less-rigid endocyclic clamps in the design of proteasome inhibitors that allow for a better presentation of residues interacting with the active site clefts of the enzyme.

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Section: Introductionmentioning

confidence: 99%