Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies.
Nanodiscs are small-sized and flat model membranes that provide a close to native environment for reconstitution of integral membrane proteins. Incorporation of membrane proteins into nanodiscs results in water-soluble proteolipid particles making the membrane proteins amenable to a multitude of bioanalytical techniques originally developed for soluble proteins. The transmembrane domain of the human CD4 receptor was fused to ubiquitin with a preceding N-terminal decahistidine tag. The resulting integral membrane protein was incorporated into nanodiscs. Binding of the nanodisc-inserted histidine-tagged protein to a monoclonal anti-pentahistidine antibody was quantified using surface plasmon resonance (SPR) experiments. For the first time, a membrane-inserted transmembrane protein was employed as analyte while the antibody served as ligand immobilized on the sensor chip surface. SPR experiments were conducted in single-cycle mode. We demonstrate that the nanodisc-incorporated membrane protein showed nearly identical affinity toward the antibody as did the soluble decahistidine-tagged ubiquitin studied in a comparative experiment. Advantages of the new experimental setup and potential applications are discussed.
Glück
Julian M
JM
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Koenig
Bernd W
BW
Willbold
Dieter
D
D000911:Antibodies, Monoclonal / Q000276:immunology; D015374:Biosensing Techniques / Q000379:methods; D015704:CD4 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049329:Nanostructures / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D008565:Membrane Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin