Atomic Force Microscopy of Proteasome Assemblies

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 736)

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.

Key words

Atomic force microscopy Single molecule analysis Proteasome Allostery Protein dynamics Enzyme catalysis Conformational selectivity 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Molecular Medicine, Institute of BiotechnologyUniversity of Texas Health Science Center at San AntonioSan AntonioUSA

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