The mechanism of Pi interaction with phosphate binding protein of Escherichia coli has been investigated using the A197C mutant protein labeled with a coumarin fluorophore (MDCC-PBP), which gives a fluorescence change on binding Pi. A pure preparation of MDCC-PBP was obtained, in which the only significant inhomogeneity is the presence of equal amounts of two diastereoisomers due to the chiral center formed on reaction of the cysteine with the maleimide. These diastereoisomers could not be separated, but Pi binding data suggest that they differ in affinity and fluorescence change. When Pi binds to MDCC-PBP, the fluorescence quantum yield increases 8-fold and the fluorescence intensity at 465 nm increases 13-fold. The kinetics of Pi binding show saturation of the rate at high Pi concentrations, and this together with other information suggests a two-step mechanism with the fluorescence change after binding, concomitant with a conformational change of the protein that closes the cleft containing the Pi binding site. Cleft closure has a rate constant of 317 s-1 (pH 7.0, 5 degrees C), and opening has a rate constant of 4.5 s-1. The fluorescence increase is likely to arise from a change in the hydrophobic environment during this closure as the steady state fluorescence emission (lambdamax and intensity) on Pi binding is mimicked by the addition of ethanol to aqueous solutions of an MDCC-thiol adduct. Fluorescence lifetimes in the absence and presence of Pi were 0.3 and 2.4 ns, respectively, consistent with the change in quantum yield. The rotational correlation time of the coumarin increases only 2-fold from 15 to 26 ns on binding Pi as measured by time-resolved polarization, consistent with the main rotation being determined by the protein even in the open conformation, but with greater local motion. Circular dichroism of the coumarin induced by the protein is weak in the absence of Pi and increases strongly upon saturation by Pi. These data are also consistent with an open to closed conformational model.

Apolipoprotein AI (apoAI) is the principal protein constituent of high density lipoproteins and it plays a key role in human cholesterol homeostasis; however, the structure of apoAI is not clearly understood. To test the hypothesis that apoAI is organized into domains, three deletion mutants of human apoAI expressed in Escherichia coli were studied in solution and in reconstituted high density lipoprotein particles. Each mutant lacked one of three specific regions that together encompass almost the entire 243 aa sequence of native apoAI (apoAI ⌬44-126, apoAI ⌬139-170, and apoAI ⌬190-243). Circular dichroism spectroscopy showed that the ␣-helical content of lipid-free apoAI ⌬44-126 was 27% while the other mutants and native apoAI averaged 55 ؎ 2%, suggesting that the missing N-terminal portion contains most of the ␣-helical structure of lipid-free apoAI. ApoAI ⌬44-126 exhibited the largest increase in ␣-helix upon lipid binding (125% increase versus an average of 25% for the others), confirming the importance of the C-terminal half of apoAI in lipid binding. Denaturation studies showed that the N-terminal half of apoAI is primarily responsible for ␣-helix stability in the lipid-free state, whereas the C terminus is required for ␣-helix stability when lipid-bound. We conclude that the N-terminal half (aa 44-126) of apoAI is responsible for most of the ␣-helical structure and the marginal stability of lipid-free apoAI while the C terminus (aa 139-243) is less organized. The increase in ␣-helical content observed when native apoAI binds lipid results from the formation of ␣-helix primarily in the C-terminal half of the molecule.

Heptahelical receptors (HHRs) are generally thought to function as monomeric entities. Several HHRs such as somatostatin receptors (SSTRs), however, form homo-and heterooligomers when activated by ligand binding. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with SSTR5 (R5) or SSTR1 (R1), or cotransfected with R5 and R1, we have analyzed the ligand receptor stoichiometry and aggregation states for the three receptor systems by fluorescence resonance energy transfer and fluorescence correlation spectroscopy. Both homo-and heterooligomeric receptors are occupied by two ligand molecules. We find that monomeric, homooligomeric, and heterooligomeric receptor species occur in the same cell cotransfected with two SSTRs, and that oligomerization of SSTRs is regulated by ligand binding by a selective process that is restricted to some (R5) but not other (R1) SSTR subtypes. We propose that induction by ligand of different oligomeric states of SSTRs represents a unique mechanism for generating signaling specificity not only within the SSTR family but more generally in the HHR family. H eptahelical receptors (HHRs) constitute the largest single family of transmembrane signaling molecules that respond to diverse external stimuli such as hormones, neurotransmitters, chemoattractants, odorants, and photons. Although these receptors have been generally thought to function as monomeric entities, there is growing evidence that a number of HHRs assemble as functional homo-and heterooligomers (1, 2). Dimerization seems to be necessary for function of the class C subfamily of HHRs comprising the metabotropic glutamate, calcium sensing, the GABAB, and pheromone receptors that are targeted to the plasma membrane as preformed dimers which are stabilized by ligand binding (3-7). Several HHRs such as somatostatin receptors (SSTRs), dopamine receptors, gonadotrophin-releasing hormone receptor (GnRHR), luteinizing hormone͞chorionic gonadotrophin hormone receptor, and chemokine receptors, however, which belong to the rhodopsin-like class A subfamily of HHRs, assemble on the membrane as homo-and heterooligomeric species in response to agonist activation (8-15).In the case of SSTRs, we have shown by photobleaching fluorescence resonance energy transfer (pbFRET) that the human (h) type 5 receptor (hSSTR5 or R5) exists in the basal state as a monomer, and that activation by ligand induces dose-dependent oligomerization (8). When coexpressed with another SSTR (hSSTR1 or R1) or an unrelated HHR such as the dopamine 2 receptor (D 2 R), R5 also forms a heterooligomer that displays pharmacological properties distinct from those of either of the separate receptors (9). Little is known about the stoichiometry of ligand-receptor reactions or the specificity for homoand heterooligomeric interactions between two receptors that are coexpressed in the same cell. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with R5 or R1, or cotransfected with R5 and R1, we have analyzed the ...

Excess light triggers protective nonradiative dissipation of excitation energy in photosystem II through the formation of a trans-thylakoid pH gradient that in turn stimulates formation of zeaxanthin and antheraxanthin. These xanthophylls when combined with protonation of antenna pigment-protein complexes may increase nonradiative dissipation and, thus, quench chlorophyll a fluorescence. Here we measured, in parallel, the chlorophyll a fluorescence lifetime and intensity to understand the-mechanism of this process. Increasing the xanthophyll concentration in the presence of a pH gradient (quenched conditions) decreases the fractional intensity of a fluorescence lifetime component centered at -2 ns and increases a component at -0.4 ns. Uncoupling the pH gradient (unquenched conditions) eliminates the 0.4-ns component. Changes in the xanthophyll concentration do not significantly affect the fluorescence lifetimes in either the quenched or unquenched sample conditions. However, there are differences in fluorescence lifetimes between the quenched and unquenched states that are due to pH-related, but nonxanthophyll-related, processes. Quenching of the maximal fluorescence intensity correlates with both the xanthophyll concentration and the fractional intensity of the 0.4-ns component. The unchanged fluorescence lifetimes and the proportional quenching ofthe maximal and dark-level fluorescence intensities indicate that the xanthophylls act on antenna, not reaction center processes. Further, the fluorescence quenching is interpreted as the combined effect of the pH gradient and xanthophyll concentration, resulting in the formation of a quenching complex with a short ("0.4 ns) fluorescence lifetime.A conserved mechanism protects photosystem II (PSII) in higher plants against damage by excess absorbed excitation energy (1, 2). It has been suggested that excess excitation of the PSII antenna is nonradiatively dissipated, causing nonphotochemical quenching (NPQ) of PSII chlorophyll (Chl) a fluorescence ( Fig. 1) (1-4). Excess light triggers NPQ by first causing chloroplast lumen acidification. Proton pumping from either light-driven electron flow or the chloroplast ATPase can induce NPQ (3). Lumen acidity serves at least two roles in the NPQ mechanism. First it activates the xanthophyll cycle deepoxidase (5) that catalyzes violaxanthin (Vx) deepoxidation to zeaxanthin (Zx) via antheraxanthin (Ax) (6). Second, protonation apparently alters the conformation of PSII antenna and/or reaction center pigment-protein complexes, promoting interaction with the xanthophylls and somehow causing NPQ (3, 4, 7). In isolated chloroplasts, NPQ depends on both lumen acidification and Zx plus Ax (4). Uncouplers of the proton gradient reverse NPQ and inhibit deepoxidation (3-5). (3,4).One proposed NPQ mechanism includes a role for Vx in preventing or reversing pH-dependent aggregation of PSII light-harvesting antenna complexes (LHCIIs) (9). Here, decreasing the Vx concentration, which is equivalent to increasing the sum of the Zx and...