Production of Disulfide-stablized Transmembrane Peptide Complexes for Structural Studies.
Journal Title
Journal of Visualized Experiments
Publication Type
Journal Article
Abstract
Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences
Publisher
MyJoVE Corporation
Research Division(s)
Structural Biology
Publisher's Version
https://doi.org/10.3791/50141
Terms of Use/Rights Notice
Copyright © JoVE 2006-2014. All Rights Reserved


Creation Date: 2014-01-14 03:36:38
An error has occurred. This application may no longer respond until reloaded. Reload 🗙