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J. Biol. Chem., Vol. 277, Issue 39, 36321-36328, September 27, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/39/36321    most recent M202145200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Thess, A. Articles by Guckenberger, R. Search for Related Content PubMed PubMed Citation Articles by Thess, A. Articles by Guckenberger, R. Social Bookmarking                      What's this?

Specific Orientation and Two-dimensional Crystallization of the Proteasome at Metal-chelating Lipid Interfaces^*

Andreas Thess^§, Silke Hutschenreiter^¶, Matthias Hofmann, Robert Tampé^¶, Wolfgang Baumeister, and Reinhard Guckenberger

From the  Abteilung Molekulare Strukturbiologie, Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany and the ^¶ Institut für Biochemie, Biozentrum, Goethe-Universität Frankfurt, Marie-Curie-Str. 9, 60439 Frankfurt a.M., Germany

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 -subunits. The N termini of -subunits might partly occlude the entrance channel in -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.

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