Specific orientation and two-dimensional crystallization of the proteasome at 
metal-chelating lipid interfaces

Thess, A.; Hutschenreiter, S.; Hofmann, M.; Tampe, R.; Baumeister, W.; Guckenberger, R.

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Specific orientation and two-dimensional crystallization of the proteasome at metal-chelating lipid interfaces

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Baumeister, W.
Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society;

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Guckenberger, R.
Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society;

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Citation

Thess, A., Hutschenreiter, S., Hofmann, M., Tampe, R., Baumeister, W., & Guckenberger, R. (2002). Specific orientation and two-dimensional crystallization of the proteasome at metal-chelating lipid interfaces. Journal of Biological Chemistry, 277(39), 36321-36328.

Cite as: https://hdl.handle.net/11858/00-001M-0000-0010-6E4C-6

Abstract

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 a-subunits might partly occlude the entrance channel in a-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.