Role of D(E)RY motif in switching mechanism of G protein-coupled receptors studied by ITC

Membrane proteins, especially GPCRs (G-protein coupled receptors), play a fundamental role in many physiological processes. Their function is based on the ability to switch between different structures. Due to the low dielectricity of the lipidic environment, side chain charges contribute to the energetics of such transitions much more than in purely aqueous environment.
Our aim is to understand the functional implication of charged amino acids in the conformational control of membrane-spanning protein domains.
We used Isothermal Titration Calorimetry (ITC) to study proton uptake from the buffer by the class-defining and highly conserved D(E)RY motif in a synthetic peptide derived from rhodopsin transmembrane (TM) helix 3. By adding a negatively charged molecule (SDS) the pKA of the peptide is expected to be shifted and proton uptake from the buffer by glutamic acid in the D(E)RY motif is assumed to occur depending on pH. Thus the heat of buffer ionization depending on pH and buffer ionization enthalpy is measured according to Baker and Murphy. First experiments in phosphate buffer at pH 4.8 showed promising results, i.e. a titration curve with stoichiometry around 1:1 (SDS:peptide) and ∆H in good agreement to buffer ionization heat of phosphate buffer. However the buffer ionization heat of phosphate buffer is comparatively low and so are other buffers at that pH. Thus these results were not sufficiently reproducible and following experiments were performed at pH 6 and 8 with Imidazole buffer, PIPES and TRIS. These results were good reproducible but no buffer ionization could be shown. A pKA shift in our model peptide introduced by an additional charge (SDS) could not be proved by means of ITC. Thus, there is no evidence for breakage of the internal Glu-Arg salt bridge by competition with an Arg-sulfate interaction.