PMID: 8804611;PMCID: PMC1233479;Abstract:
Surface plasmon resonance (SPR) spectroscopy can provide useful information regarding average structural properties of membrane films supported on planar solid substrates. Here we have used SPR spectroscopy for the first time to monitor the binding and activation of G-protein (transducin or G(t)) by bovine rhodopsin incorporated into an egg phosphatidylcholine bilayer deposited on a silver film. Rhodopsin incorporation into the membrane, performed by dilution of a detergent solution of the protein, proceeds in a saturable manner. Before photolysis, the SPR data show that G(t) binds tightly (K(eq) ≃ 60 nM) and with positive cooperativity to rhodopsin in the lipid layer to form a closely packed film. A simple multilayer model yields a calculated average thickness of about 57 .Å, in good agreement with the structure of G(t). The data also demonstrate that G(t) binding saturates at a G(t)/rhodopsin ratio of approximately 0.6. Moreover, upon visible light irradiation, characteristic changes occur in the SPR spectrum, which can be modeled by a 6 Å increase in the average thickness of the lipid/protein film caused by formation of metarhodopsin II (MII). Upon subsequent addition of GTP, further SPR spectral changes are induced. These are interpreted as resulting from dissociation of the α- subunit of G(t), formation of new MII-G(t) complexes, and possible conformational changes of G(t) as a consequence of complex formation. The above results clearly demonstrate the ability of SPR spectroscopy to monitor interactions among the proteins associated with signal transduction in membrane-bound systems.
Well-resolved proton (1H) NMR spectra of solid human arterial plaque can be acquired. Studies have been carried out of human fatty plaque obtained postmortem (ex vivo), the total lipids extracted from human atheroma, and a model mixture of cholesteryl esters whose lipid composition resembles that of human atheroma. In each case, well-resolved 1H NMR spectra were obtained at body temperature (37°C), with little or no underlying broad signal. Such sharp 1H NMR spectra are typical of isotropic fluids, whereas solid and liquid-crystalline materials give rise to much broader spectral lines. The results suggest the sharp 1H NMR spectra of human atheromatous lesions at body temperature are due largely to the presence of intracellular and extracellular droplets of cholesteryl esters in the isotropic liquid phase. These findings provide a necessary basis for use of 1H NMR techniques to image quantitatively the lipid constituents of human atheroma in vivo, and to study their chemical and physical properties. © 1987.
Polyunsaturated phospholipids of the omega-3 and omega-6 classes play key roles in cellular functions, yet their mechanisms of biological action are still a matter of debate. Using deuterium ((2)H) NMR spectroscopy and small-angle X-ray diffraction, we show how membrane properties are modified by docosahexaenoic (DHA; 22:6) and arachidonic (AA; 20:4) acyl chains of the omega-3 and the omega-6 families, respectively. Structural and dynamical differences due to polyunsaturation are evident in both the ordered and disordered phases of mixed-chain (16:0)(22:6)PC and (16:0)(20:4)PC bilayers. Due to the lower chain melting temperature, the omega-6 AA bilayer is more disordered in the fluid (L(alpha)) state than the omega-3 DHA bilayer; it is thinner with a larger area per lipid. The thermal hysteresis observed for the DHA bilayer may represent the influences of angle-iron conformers in the gel state and back-bended, hairpinlike conformers in the fluid state, consistent with molecular dynamics studies. Interpretation of the (2)H NMR order profiles of (16:0-d(31))(22:6)PC and (16:0-d(31))(20:4)PC together with X-ray electron density profiles reveals an uneven distribution of mass; i.e., the sn-1 saturated chain is displaced toward the membrane center, whereas the sn-2 polyunsaturated chain is shifted toward the bilayer aqueous interface. Moreover, the (2)H NMR relaxation rates are increased by the presence of omega-6 AA chains compared to omega-3 DHA chains. When evaluated at the same amplitude of motion, relaxation parameters give a naturally calibrated scale for comparison of fluid lipid bilayers. Within this framework, polyunsaturated bilayers are relatively soft to bending and area fluctuations on the mesoscale approaching molecular dimensions. Significant differences are evident in the viscoelastic properties of the omega-3 and omega-6 bilayers, a possibly biologically relevant feature that distinguishes between the two phospholipid classes.