Heterotrimeric G protein β 1 γ 2 subunits change orientation upon complex formation with G protein-coupled receptor kinase 2 (GRK2) on a model membrane

Few experimental techniques can assess the orientation of peripheral membrane proteins in their native environment. Sum Frequency Generation (SFG) vibrational spectroscopy was applied to study the formation of the complex between G protein-coupled receptor (GPCR) kinase 2 (GRK2) and heterotrimeric G protein β 1 γ 2 subunits (Gβγ) at a lipid bilayer, without any exogenous labels. The most likely membrane orientation of the GRK2-Gβγ complex differs from that predicted from the known protein crystal structure, and positions the predicted receptor docking site of GRK2 such that it would more optimally interact with GPCRs. Gβγ also appears to change its orientation after binding to GRK2. The developed methodology is widely applicable for the study of other membrane proteins in situ. With the addition of more measurements from other techniques, we believe that it will be possible to assess the exact orientation of proteins that interact with biological membranes, and how the binding of additional components affects the orientation, and hence the function, of these complexes. Such studies would improve our understanding of the molecular basis of a variety of cellular processes, particularly those involving the recruitment of signaling molecules to the cell surface. For example, formation of the GRK2-Gβγ complex is thought to play an important role in the progression of heart failure and cardiac hypertrophy. The results obtained in this study represent another step towards a complete understanding of the molecular mechanisms underlying signal transduction at biological interfaces. The tools and methods developed in this research are widely applicable to the analysis of other membrane protein complexes. SFG signals were collected from the proteins associated with the lipid bilayer ( Fig. P1 ) in the so-called Amide I spectral region, which can provide a guide to the overall orientation of a protein backbone. Spectra of the GRK2-Gβγ complex were markedly different from those of the Gβγ subunit alone, and it was shown that the complex could be formed in situ. To assess the orientation of the large GRK2-Gβγ protein complex, the single experimentally observed quantity ( , the ratio of signals from two laser polarizations) was not sufficient. An additional measurement was obtained, based on the observation that the polarized SFG signal intensity from the Gβγ subunit alone was greater than the signal from the entire GRK2-Gβγ complex, which is only possible for certain or