title: Unveiling the membrane-binding properties of N-terminal and C-terminal regions of G protein-coupled receptor kinase 5 by combined optical spectroscopies.
abstract: G protein-coupled receptor kinase 5 (GRK5) is thought to associate with membranes in part via N- and C-terminal segments that are typically disordered in available high-resolution crystal structures. Herein we investigate the interactions of these regions with model cell membrane using combined sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. It was found that both regions associate with POPC lipid bilayers but adopt different structures when doing so: GRK5 residues 2-31 (GRK5(2-31)) was in random coil whereas GRK5(546-565) was partially helical. When the subphase for the GRK5(2-31) peptide was changed to 40% TFE/60% 10 mM phosphate pH 7.4 buffer, a large change in the SFG amide I signal indicated that GRK5(2-31) became partially helical. By inspecting the membrane behavior of two different segments of GRK5(2-31), namely, GRK5(2-24) and GRK5(25-31), we found that residues 25-31 are responsible for membrane binding, whereas the helical character is imparted by residues 2-24. With SFG, we deduced that the orientation angle of the helical segment of GRK5(2-31) is 46 ± 1° relative to the surface normal in 40% TFE/60% 10 mM phosphate pH = 7.4 buffer but increases to 78 ± 11° with higher ionic strength. We also investigated the effect of PIP2 in the model membrane and concluded that the POPC:PIP2 (9:1) lipid bilayer did not change the behavior of either peptide compared to a pure POPC lipid bilayer. With ATR-FTIR, we also found that Ca(2+)·calmodulin is able to extract both peptides from the POPC lipid bilayer, consistent with the role of this protein in disrupting GRK5 interactions with the plasma membrane in cells.
authors.affiliation: Department of Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Bei
authors.initials: B
authors.lastname: Ding
authors.forename: Alisa
authors.initials: A
authors.lastname: Glukhova
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Sobczyk-Kojiro
authors.forename: Henry I
authors.initials: HI
authors.lastname: Mosberg
authors.forename: John J G
authors.initials: JJ
authors.lastname: Tesmer
authors.forename: Zhan
authors.initials: Z
authors.lastname: Chen
mesh_terms: D001665:Binding Sites; D054774:G-Protein-Coupled Receptor Kinase 5 / Q000138:chemical synthesis / Q000737:chemistry; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry*; D013057:Spectrum Analysis
chemical_list: D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D054774:G-Protein-Coupled Receptor Kinase 5; C517898:GRK5 protein, human; C028694:1-palmitoyl-2-oleoylphosphatidylcholine