Unveiling the membrane-binding properties of N-terminal and C-terminal regions of G protein-coupled receptor kinase 5 by combined optical spectroscopies

Bei Ding, Alisa Glukhova, Katarzyna Sobczyk-Kojiro, Henry I. Mosberg, John J G Tesmer, Zhan Chen

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4 Citations (Scopus)

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 (GRK52-31) was in random coil whereas GRK5546-565 was partially helical. When the subphase for the GRK52-31 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 GRK52-31, namely, GRK52-24 and GRK525-31, 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 Ca2+·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. 

Original languageEnglish
Pages (from-to)823-831
Number of pages9
JournalLangmuir
Volume30
Issue number3
DOIs
Publication statusPublished - 28 Jan 2014
Externally publishedYes

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