G protein-coupled receptors (GPCRs) recognize a wide variety of extracellular ligands to control diverse physiological processes. Compounds that bind to such receptors can either stimulate, fully or partially (full or partial agonists), or reduce (inverse agonists) the receptors' basal activity and receptor-mediated signaling. Various studies have shown that the activation of receptors through binding of agonists proceeds by conformational changes as the receptor switches from a resting to an active state leading to G protein signaling. Yet the molecular basis for differences between agonists and inverse agonists is unclear. These different classes of compounds are assumed to switch the receptors' conformation in distinct ways. It is not known, however, whether such switching occurs along a linear 'on-off' scale or whether agonists and inverse agonists induce different switch mechanisms. Using a fluorescence-based approach to study the alpha2A-adrenergic receptor (alpha(2A)AR), we show that inverse agonists are differentiated from agonists in that they trigger a very distinct mode of a receptor's switch. This switch couples inverse agonist binding to the suppression of activity in the receptor.

Reversible photoinduced reorientations of bacteriorhodopsin have been detected in suspensions of the purple membrane of Halobacterium salinarium. The anisotropy in bacteriorhodopsin during the nanosecond through millisecond stages of the photocycle was measured by time-resolved linear dichroism and transient absorption measurements. From these measurements the anisotropies of the K, L, M, and O intermediates were determined and related to the chromophore orientation with respect to the initially selected orientation. The anisotropies of the K and L states are 0.38 +/- 0.01 and 0.35 +/- 0.01, respectively. Further anisotropy decay after formation of the M intermediate in about 0.5 ms is evidence of orientational motion at this stage in the photocycle. A constant anisotropy with a value of 0.39 +/- 0.02 in the O intermediate demonstrates a recovery of the initial protein orientation with the formation of the O state. These results demonstrate that reorientations in BR are photoinduced and reversible. Similar measurements for L and M were carried out for purple membrane in polyacrylamide gels, where the anisotropies in the L and M states are 0.38 +/- 0.014 and 0.36 +/- 0.01, respectively. These results show that reorientations also occur in BR immobilized in gels. Anisotropy decay in the M state after formation of the M intermediate was not detected in the gels, in contrast to the M intermediate in suspensions. Orientational changes are observed for BR in purple membrane suspensions in the K state, during the K-->L step, in the M state possibly related to an M1-->M2 transition, and in the O state, where an almost complete return to the original orientation occurs.(ABSTRACT TRUNCATED AT 250 WORDS)

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Aggregation is an important area of scientific investigation because of the consequences of this process for many aspects of protein and peptide chemistry. Previous studies of the aggregation of the βA4 peptide fragment, β(12−28), and synthetic analogues in low pH aqueous solution show that replacing either or both phenylalanines with glycine reduces the tendency of this peptide to form aggregates. In this investigation, several β(12−28) analogues have been synthesized in which the phenylalanine residues 19 and/or 20 have been substituted with the nonnative amino acid, naphthylalanine, to produce the peptides [napAla19,20], [napAla19,Gly20], and [Gly19,napAla20] and allowing the aggregation behavior of these peptides to be examined in aqueous solution at low pH with both NMR and fluorescence spectroscopy. The NMR chemical shift, diffusion coefficients and relaxation times as well as rotational correlation times measured with both NMR and fluorescence spectroscopy are concentration dependent providing evidence that [napAla19,20]β(12−28) forms soluble aggregates. Similar results obtained for [napAla19,Gly20]β(12−28) and [Gly19,napAla20]β(12−28) suggest that these peptides have a greatly reduced tendency to aggregate. In addition, [napAla19,20]β(12−28) produces excimer fluorescence emission in a concentration-dependent manner with essentially no excimer detected in the fluorescence spectra of the singly substituted naphthylalanine analogues. Fluorescence lifetimes were measured, and unlike naphthylalanine, the free amino acid, the excimer fluorescence decay of [napAla19,20]β(12−28) does not exhibit a rise time component, suggesting a ground-state preassociation of the peptides through naphthyl π−π interactions that stabilize the aggregates. Fluorescence spectroscopy, due to its concentration sensitivity, permits measurements of peptide solutions at much lower concentration than NMR, allowing direct measurement of the peptide monomer. However, NMR spectroscopy, through the measurement of nuclear relaxation times, can provide complementary information about the differential regional mobility of the peptide. The application of both NMR and fluorescence spectroscopy to the analysis of these naphthyl-substituted peptides produces a more complete picture of their aggregation behavior than could be obtained using either method alone. An advantage of using the combination of these methods is that their different time scales make them sensitive to different ranges of molecular motion.

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