Specific orientation and two-dimensional crystallization of the proteasome at metal-chelating lipid interfaces.
The potential of a protein-engineered His tag to immobilize macromolecules in a predictable orientation at metal-chelating lipid interfaces was investigated using recombinant 20 S proteasomes His-tagged in various positions. Electron micrographs demonstrated that the orientation of proteasomes bound to chelating lipid films could be controlled via the location of their His tags: proteasomes His-tagged at their sides displayed exclusively side-on views, while proteasomes His-tagged at their ends displayed exclusively end-on views. The activity of proteasomes immobilized at chelating lipid interfaces was well preserved. In solution, His-tagged proteasomes hydrolyzed casein at rates comparable with wild-type proteasomes, unless the His tags were located in the vicinity of the N termini of alpha-subunits. The N termini of alpha-subunits might partly occlude the entrance channel in alpha-rings through which substrates enter the proteasome for subsequent degradation. A combination of electron micrographs and atomic force microscope topographs revealed a propensity of vertically oriented proteasomes to crystallize in two dimensions on fluid lipid films. The oriented immobilization of His-tagged proteins at biocompatible lipid interfaces will assist structural studies as well as the investigation of biomolecular interaction via a wide variety of surface-sensitive techniques including single-molecule analysis.
Abteilung Molekulare Strukturbiologie, Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany.
Andreas
A
Thess
Silke
S
Hutschenreiter
Matthias
M
Hofmann
Robert
R
Tampé
Wolfgang
W
Baumeister
Reinhard
R
Guckenberger
D000327:Adsorption; D002614:Chelating Agents / Q000494:pharmacology*; D003460:Crystallization; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D050356:Lipid Metabolism; D008670:Metals / Q000494:pharmacology*; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary; D013822:Thermoplasma / Q000378:metabolism
D002614:Chelating Agents; D008051:Lipid Bilayers; D008670:Metals; D009097:Multienzyme Complexes; D006639:Histidine; D009532:Nickel; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex