Weak alignment of membrane proteins in stressed polyacrylamide gels.
Residual dipolar couplings are important as angular constraints for the structure determination of membrane proteins in micelles. Strained polyacrylamide gels are one of the few available mechanisms available for inducing the requisite weak alignment for these samples. However, their use is frequently limited by the ability to incorporate proteins and buffer solutions into the gel matrix. The implementation of several methods of incorporating membrane proteins into gels are described. Conditions for copolymerizing the protein in the absence of a change in pH are detailed. Electrophoresis is also shown to be a useful method to incorporate proteins. Weak alignment of the protein-micelle complex in the gel matrix is subsequently achieved using either vertical or radial compression. The magnitude of alignment can be controlled by altering the gel concentration, the acrylamide/bisacrylamide ratio, and the compression ratio. The alignment tensor can be altered relative to uncharged polyacrylamide gels by copolymerizing samples with acrylamide/acrylic acid to incorporate negative charges in the strained polyacrylamide gel to provide an alternate orientation.
Jones
David H
DH
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA.
Opella
Stanley J
SJ
D000180:Acrylic Resins; D036022:Capsid Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*
D000180:Acrylic Resins; D036022:Capsid Proteins; D008565:Membrane Proteins; D008823:Micelles; D009587:Nitrogen Isotopes; C119217:coat protein pVIII, Enterobacteria phage fd; C016680:polyacrylamide gels