Solution NMR Spectroscopy and Protein Interaction Studies of Membrane Proteins in Nanodiscs

Abstract

Insolubility of integral membrane proteins (IMPs) in buffer often prevents their investigation with biophysical methods developed for soluble proteins. First, solution NMR of large proteins or complexes is limited by slow rotational diffusion. IMPs in cellular membranes or liposomes are beyond the size limit of solution NMR. Therefore, detergent micelles or bicelles are often used for solubilization and NMR studies of IMPs. Unfortunately, detergents may destabilize the native structure or compromise the activity of proteins. Nanodiscs are a lipid-based, detergent free, relatively small and very promising new model membrane system. We inserted a recombinantly produced and 15N, 13C-labeled fragment of human CD4 into nanodiscs. This polypeptide comprises the membrane-spanning and the cytoplasmic domains of CD4. Our NMR data demonstrate the feasibility of solution NMR spectroscopy on IMPs in nanodiscs. Second, surface plasmon resonance (SPR) is predominantly utilized in interaction studies of soluble proteins. Incorporation of IMPs into nanodiscs provides a close to native environment to the membrane protein and results in a water-soluble proteolipid particle that might be amenable to standard SPR-based methodology. We reconstituted a decahistidine-tagged IMP into nanodiscs and studied binding between the nanodisc-inserted IMP and a PentaHis monoclonal antibody (mAb) immobilized on the surface of a CM5-sensorchip. For comparison, we also determined the affinity of the decahistidine-tagged soluble domain of the same IMP toward the immobilized PentaHis mAb. Binding affinities were almost identical in both cases. However, the association and dissociation rate constants were found to differ, which is in agreement with the distinct diffusion coefficients of the soluble analyte particles. Our data indicate that nanodisc-inserted IMPs can serve as analyte in interaction studies of membrane proteins.