title: Purification of Protein Chaperones and Their Functional Assays with Intermediate Filaments.
abstract: Intermediate filament (IF) scaffolds facilitate small heat shock protein (sHSP) function, while IF function is sHSP dependent. sHSPs interact with IFs and the importance of this interaction is to maintain the individuality of the IFs and to modulate interfilament interactions both in networks and in assembly intermediates. Mutations in both sHSPs and their interacting IF proteins phenocopy each other in the human diseases they cause. This establishes a key functional relationship between these two very distinct protein families, and it also evidences the role of this cytoskeleton-chaperone complex in the cellular stress response. In this chapter, we describe the detailed experimental protocols for the preparation of purified IF proteins and sHSPs to facilitate the study in vitro of their functional interactions. In addition, we describe the detailed biochemical procedures to assess the effect of sHSP on the assembly of IFs, the binding to IFs, and the prevention of noncovalent filament-filament interactions using in vitro cosedimentation, electron microscopy, and viscosity assays. These assays are valuable research tools to study and manipulate sHSP-IF complexes in vitro and therefore to determine the structure-function detail of this complex, and how it contributes to cellular, tissue, and organismal homeostasis and the in vivo stress response.
authors.affiliation: Institute of Molecular Medicine, College of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan. Electronic address: mdperng@life.nthu.edu.tw.
authors.forename: Ming-Der
authors.initials: MD
authors.lastname: Perng
authors.affiliation: Institute of Molecular Medicine, College of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan.
authors.forename: Yu-Shan
authors.initials: YS
authors.lastname: Huang
authors.affiliation: Biophysical Sciences Institute, University of Durham, Durham, United Kingdom.
authors.forename: Roy A
authors.initials: RA
authors.lastname: Quinlan
mesh_terms: D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D007382:Intermediate Filaments / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D003598:Cytoskeletal Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins
keywords: CRYAB; Cardiomyopathy; Desmin; GFAP; Proteinopathy; Proteostasis; Vimentin; αB-crystallin