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

Download full text files

Export metadata

Author:Andreas ThessORCiD, Silke HutschenreiterGND, Matthias HofmannGND, Robert TampéORCiDGND, Wolfgang BaumeisterORCiDGND, Reinhard Guckenberger
Parent Title (English):Journal of biological chemistry
Publisher:American Society for Biochemistry and Molecular Biology Publications
Place of publication:Bethesda, Md
Document Type:Article
Date of Publication (online):2021/01/04
Year of first Publication:2002
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2023/11/09
Page Number:8
First Page:36321
Last Page:36328
Institutes:Biochemie, Chemie und Pharmazie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Licence (German):License LogoCreative Commons - Namensnennung 4.0