title: Purification and Structural Analysis of Plectin and BPAG1e.
abstract: Plectin and BPAG1e belong to the plakin family of high-molecular-weight proteins that interconnect the cytoskeletal systems and anchor them to junctional complexes. Plectin and BPAG1e are prototypical plakins with a similar tripartite modular structure. The N- and C-terminal regions are built of multiple discrete structural domains, while the central rod domain mediates dimerization by coiled-coil interactions. Owing to the mosaic organization of plakins, the structure of their constituent individual domains or small multi-domain segments can be analyzed isolated. Yet, understanding the integrated function of large regions, oligomers, and heterocomplexes of plakins is difficult due to the large and segmented structure. Here, we describe methods for the production of plectin and BPAG1e samples suitable for structural and biophysical analysis. In addition, we discuss the combination of hybrid methods that yield information at several resolution levels to study the complex, multi-domain, and flexible structure of plakins.
authors.affiliation: Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas, University of Salamanca, Salamanca, Spain.
authors.forename: José A
authors.initials: JA
authors.lastname: Manso
authors.affiliation: Centro Universitario de la Defensa, Academia General Militar, Zaragoza, Spain.
authors.forename: Inés
authors.initials: I
authors.lastname: García Rubio
authors.affiliation: Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas, University of Salamanca, Salamanca, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Gómez-Hernández
authors.affiliation: Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas, University of Salamanca, Salamanca, Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: Ortega
authors.affiliation: Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas, University of Salamanca, Salamanca, Spain; Metabolic Engineering Group, Department of Microbiology and Genetics, University of Salamanca, Salamanca, Spain.
authors.forename: Rubén M
authors.initials: RM
authors.lastname: Buey
authors.affiliation: Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas, University of Salamanca, Salamanca, Spain.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Carballido
authors.affiliation: Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas, University of Salamanca, Salamanca, Spain.
authors.forename: Arturo
authors.initials: A
authors.lastname: Carabias
authors.affiliation: Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas, University of Salamanca, Salamanca, Spain.
authors.forename: Noelia
authors.initials: N
authors.lastname: Alonso-García
authors.affiliation: Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas, University of Salamanca, Salamanca, Spain. Electronic address: pereda@usal.es.
authors.forename: José M
authors.initials: JM
authors.lastname: de Pereda
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D018360:Crystallography, X-Ray; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000302:isolation & purification*; D000071821:Dystonin; D004926:Escherichia coli; D006801:Humans; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D051190:Plectin / Q000737:chemistry / Q000302:isolation & purification*; D017433:Protein Structure, Secondary; D053838:Scattering, Small Angle
chemical_list: D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C486268:DST protein, human; D000071821:Dystonin; D009419:Nerve Tissue Proteins; C495332:PLEC protein, human; D010446:Peptide Fragments; D051190:Plectin
keywords: Crystallography; DEER; Hybrid methods; Plakin; Protein structure; Recombinant expression; SAXS; Spectrin repeat