ABSTRACT
In order to monitor membrane-protein binding in lipid bilayers at physiological protein concentrations, we employed the recently developed dual-focus fluorescence correlation spectroscopy (2fFCS) technique. In a case study on a photoreceptor consisting of seven transmembrane helices and its cognate transducer (two transmembrane helices), the lateral diffusion for these integral membrane proteins was analyzed in giant unilamellar vesicles (GUVs). The two-dimensional diffusion coefficients of both separately diffusing proteins differ significantly, with D = 2.2 x 10(-8) cm2 s(-1) for the photoreceptor and with D = 4.1 x 10(-8) cm2 s(-1) for the transducer. In GUVs with both membrane proteins present together, we observed significantly smaller diffusion coefficients for labelled transducer molecules; this indicates the presence of larger diffusing units and therefore intermolecular protein binding. Based on the phenomenological dependence of diffusion coefficients on the molecule's cylindrical radius, we are able to estimate the degree of membrane protein binding on a quantitative level.
Subject(s)
Archaeal Proteins/chemistry , Carotenoids/chemistry , Photoreceptors, Microbial/chemistry , Unilamellar Liposomes/chemistry , Archaeal Proteins/metabolism , Carotenoids/metabolism , Diffusion , Lipid Bilayers/chemistry , Membrane Proteins/chemistry , Photoreceptors, Microbial/metabolism , Protein Binding , Spectrometry, FluorescenceABSTRACT
Lipid-protein interactions are known to play a crucial role in structure and physiological activity of integral membrane proteins. However, current technology for membrane protein purification necessitates extraction from the membrane into detergent micelles. Also, due to experimental protocols, most of the data available for membrane proteins is obtained using detergent-solubilized samples. Stable solubilization of membrane proteins is therefore an important issue in biotechnology as well as in biochemistry and structural biology. An understanding of solubilization effects on structural and functional properties of specific proteins is of utmost relevance for the evaluation and interpretation of experimental results. In this study, a comparison of structural and kinetic data obtained for the archaebacterial photoreceptor/transducer complex from Natronomonas pharaonis (NpSRII/NpHtrII) in detergent-solubilized and lipid-reconstituted states is presented. Laser flash photolysis, fluorescence spectroscopy, and electron paramagnetic resonance spectroscopy data reveal considerable influence of solubilization on the photocycle kinetics of the receptor protein and on the structure of the transducer protein. Especially the protein-membrane proximal region and the protein-protein interfacial domains are sensitive towards non-native conditions. These data demonstrate that relevance of biochemical and structural information obtained from solubilized membrane proteins or membrane protein complexes has to be evaluated carefully.
