Sensory rhodopsin II/transducer complex formation in detergent and in lipid bilayers studied with FRET |
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Authors: | J Kriegsmann JP Klare M Engelhard |
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Institution: | a Forschungszentrum Jülich, INB-2, Biologische Strukturforschung, D-52425 Jülich, Germany b Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany |
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Abstract: | The photophobic receptor from Natronomonas pharaonis (NpSRII) forms a photo-signalling complex with its cognate transducer (NpHtrII). In order to elucidate the complex formation in more detail, we have studied the intermolecular binding of both constituents (NpSRII and NpHtrII157; truncated at residue 157) in detergent buffers, and in lipid bilayers using FRET. The data for hetero-dimer formation of NpSRII/NpHtrII in detergent agrees well with KD values (∼ 200 nM) described in the literature. In lipid bilayers, the binding affinity between proteins in the NpSRII/NpHtrII complex is at least one order of magnitude stronger. In detergent the strength of binding is similar for both homo-dimers (NpSRII/NpSRII and NpHtrII/NpHtrII) but significantly weaker (KD ∼ 16 μM) when compared to the hetero-dimer. The intermolecular binding is again considerably stronger in lipid bilayers; however, it is not as strong as that observed for the hetero-dimer. At a molar transducer/lipid ratio of 1:2000, which is still well above physiological concentrations, only 40% homo-dimers are formed. Apparently, in cell membranes the formation of the assumed functionally active oligomeric 2:2 complex depends on the full-length transducer including the helical cytoplasmic part, which is thought to tighten the transducer-dimer association. |
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Keywords: | FRET Fö rster resonance energy transfer NpSRII Natronomonas pharaonis sensory rhodopsin II NpHtrII157 Natronomonas pharaonis transducer of sensory rhodopsin II truncated at residue 157 DDM β-dodecyl-D-maltoside POPC palmitoyl oleoyl phosphatidyl choline LUV large lamellar vesicle QY fluorescence quantum yield ITC isothermal titration calorimetry EPR electron paramagnetic resonance |
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