Atomic force microscopy of proteasome assemblies.

Maria E Gaczynska, Pawel Osmulski

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Original languageEnglish
Pages (from-to)117-132
Number of pages16
JournalMethods in molecular biology (Clifton, N.J.)
Volume736
DOIs
StatePublished - 2011

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Atomic Force Microscopy
Proteasome Endopeptidase Complex
Proteins
Multifunctional Enzymes
Allosteric Regulation
Molecular Dynamics Simulation
Ubiquitin
Eukaryota
Catalysis
Proteolysis
Names
Catalytic Domain
Electron Microscopy
Peptide Hydrolases
Magnetic Resonance Spectroscopy
Yeasts
Ligands
Mutation

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Atomic force microscopy of proteasome assemblies. / Gaczynska, Maria E; Osmulski, Pawel.

In: Methods in molecular biology (Clifton, N.J.), Vol. 736, 2011, p. 117-132.

Research output: Contribution to journalArticle

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abstract = "The proteasome is the essential prime protease in all eukaryotes. The large, multisubunit, modular, and multifunctional enzyme is responsible for the majority of regulated intracellular protein degradation. It constitutes a part of the multienzyme ubiquitin-proteasome pathway, which is broadly implicated in recognition, tagging, and cleavage of proteins. The name {"}proteasome{"} refers to several types of protein assemblies sharing a common catalytic core particle. Additional protein modules attach to the core, regulate its activities, and broaden its functional capabilities. The structure of proteasomes has been studied extensively with multiple methods. The crystal structure of the core particle was solved for several species. However, only a single structure of the core particle decorated with PA26 activator has been determined. NMR spectroscopy was successfully applied to probe a much -simpler, archaebacterial type of the core particle. In turn, electron microscopy was very effective in exploring the spatial arrangement of many classes of assemblies. Still, the makeup of higher-order -complexes is not well established. Besides, the crystal structure provided very limited information on proteasome molecular dynamics. Atomic force microscopy (AFM) is an ideal technique to address questions that are unanswered by other approaches. For example, AFM is perfectly suited to study allosteric regulation of proteasome, the role of protein dynamics in enzymatic catalysis, and the spatial organization of modules and subunits in assemblies. Here, we present a method that probes the conformational diversity and dynamics of yeast core particle using the oscillating mode AFM in liquid. We are taking advantage of the observation that the tube-shaped core particle is equipped with a swinging gate leading to the catalytic chamber. We demonstrate how to identify distinct gate conformations in AFM images and how to characterize the gate dynamics controlled with ligands and disturbed by mutations.",
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