Purification and Structural Analysis of Plectin and BPAG1e

Methods Enzymol. 2016;569:177-96. doi: 10.1016/bs.mie.2015.05.002. Epub 2015 May 29.

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.

Keywords: Crystallography; DEER; Hybrid methods; Plakin; Protein structure; Recombinant expression; SAXS; Spectrin repeat.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carrier Proteins / chemistry
  • Carrier Proteins / isolation & purification*
  • Crystallography, X-Ray
  • Cytoskeletal Proteins / chemistry
  • Cytoskeletal Proteins / isolation & purification*
  • Dystonin
  • Escherichia coli
  • Humans
  • Models, Molecular
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / isolation & purification*
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Plectin / chemistry
  • Plectin / isolation & purification*
  • Protein Structure, Secondary
  • Scattering, Small Angle

Substances

  • Carrier Proteins
  • Cytoskeletal Proteins
  • DST protein, human
  • Dystonin
  • Nerve Tissue Proteins
  • PLEC protein, human
  • Peptide Fragments
  • Plectin