Single assemblies of the intact light-harvesting complex LH2 from Rhodopseudomonas acidophila were bound to mica surfaces at 300 K and examined by observing their fluorescence after polarized light excitation. The complexes are generally not cylindrically symmetric. They act like elliptic absorbers, indicating that the high symmetry found in crystals of LH2 is not present when the molecules are immobilized on mica. The ellipticity and the principal axes of the ellipses fluctuate on the time scale of seconds, indicating that there is a mobile structural deformation. The B850 ring of cofactors shows significantly less asymmetry than B800. The photobleaching strongly depends on the presence of oxygen.The light-harvesting complexes are essential to photosynthesis in plants and bacteria. They absorb light from the sun and efficiently transport the photon energy to chemically reactive centers. The spectroscopic properties of the complex LH2 from photosynthetic bacteria, Rhodopseudomonas acidophila strain 10050, whose structure at atomic resolution was determined by Cogdell and coworkers (1), have been reviewed recently (2). The complex is notable for its high symmetry arrangement of the nine ␣␤-dipeptides that form the scaffold that holds the associated bacteriochlorophyll (Bchl) cofactors in place. These cofactors form into two rings that have approximate 9-fold rotation symmetry. The B800 ring consists of nine monomeric Bchls located, peripherally, between the ␤-apoproteins and closest to the N-terminal ends, whereas B850 consists of nine pairs of Bchls each associated with one ␣␤-dipeptide (1). Light absorbed in the B800 ring is transferred to B850 in less than a picosecond. In vivo the energy is transferred from B850 to the complex LH1, which also has high symmetry, and from there to a reaction center where charge separation occurs. Properties of excitations in extended systems depend on the interplay between the nuclear motions that tend to localize excitations, and the delocalizing effect of the interaction between the cofactors (3). Therefore the nature of the excitations present in the LH1 and LH2 complexes must depend not only on the static or average structures but also on the structural fluctuations that occur in solution at ambient temperature.Single molecule methods are well suited for the examination of the slow structural fluctuations (4), which are representative of the rough energy landscapes of macromolecules. Fluctuations occurring on the microsecond to minutes time scales would be manifest in conventional bulk spectroscopies as a quasistatic inhomogeneous broadening. Single macromolecules can be studied by means of fluorescent probes. Fluctuations of the macromolecular structure and orientation can result in intensity, lifetime and spectral changes of the probe fluorescence. Such methods were used to examine protein dynamics (5-7), enzyme reactions (8, 9) and nucleic acid motions (10, 11). In the case of the light-harvesting complexes a fluorescent probe is unnecessary because the intrinsic...

A comparative study is presented of competitive fluorescences of three flavonols, 3-hydroxyflavone, 3,3',4',7-tetrahydroxyflavone (fisetin), and 4'-diethylamino-3-hydroxyflavone (DEIF). The normal fluorescence Si -* So (400-nm region) is largely replaced by the proton-transfer tautomer fluorescence S' --S' in the 550-nm region for all three of the flavonols in aprotic solvents at room temperature. For DHF in polar solvents the normal fluorescence becomes a charge-transfer fluorescence (460-500 nm) which competes strongly with the still dominant proton-transfer fluorescence (at 570 nm). In protic solvents, and at 77 K, the interference with intramolecular hydrogen bonding gives rise to greatly enhanced normal fluorescence, lowering the quantum yield of proton-transfer fluorescence. The utility of DHF as a discriminating fluorescence probe for protein binding sites is suggested by the strong dependence of the charge-transfer fluorescence on polarity of the environment and by various static and dynamic parameters of the charge-transfer and protontransfer fluorescence which can be determined.The purpose of this paper is to explore the application of molecular fluorescence as probes for protein binding sites. The competition between proton-transfer (PT) fluorescence (1, 2) (i.e., excited-state intramolecular proton transfer, ESIPT) and charge-transfer (CT) fluorescence, and the application of solvent polarity studies, is presented for three hydroxyflavones selected as fluorescence probes. The model compounds chosen are 3-hydroxyflavone (3-HF) and its derivatives 3,3',4',7-tetrahydroxyflavone (fisetin) and 4'-diethylamino-3-hydroxyflavone (DHF). In the first two, PT tautomer fluorescence (Amax 529 nm and 540 nm, respectively) is predominant in aprotic solvents at 298 K, the normal tautomer fluorescence being observed only very weakly. The third molecule (DHF) emits both a PT fluorescence at Amos 570 nm and a prominent CT fluorescence at Amass 460 nm.3-HF has been the object of extensive photophysical studies (1-33). Fisetin has not been studied extensively (29) and attracted our attention again recently because of its extraordinary performance as a lasing material (unpublished work). DHF is a newly synthesized molecule (34-36) which we explore as a protein fluorescence probe.The spectroscopic behavior of these three hydroxyflavones offers some contrasting properties which, for these cases, permit a discrimination between normal fluorescence, strong CT fluorescence, and PT fluorescence and indicate the utility of the latter two as fluorescence probe phenomena. MATERIALS AND METHODSSpectroscopic Measurements. Absorption spectra were measured on a Shimadzu UV-2100 spectrophotometer. The fluorescence spectra were recorded with a SPEX Fluoromax spectrofluorimeter (Spex Industries, Edison, NJ).Chemicals. 3-HF and fisetin were from Aldrich. tDHF and 4'-diethylamino-3-methoxyflavone were synthesized in the laboratory of Pi-Tai Chou (University of South Carolina, Columbia). All solvents were of spectrograde quality and...

A fluorescence probe is introduced for protein conformation and binding-site monitoring as the protontransfer (PT) tautomer fluorescence by using 4-hydroxy-5-azeanthrene (HAP) as a prototype. A typical grosslywavelength-shifted PT fluorescence for HAP is observed in the 600-nm spectral region for this UV-absorbing moeule (absorption onset, 400 um), for which case PT occurs even in protic solvents. It is shown that PT fluorescence of HAP can serve as a protein-binding-site static-polarity calibrator, fig frm a A.,. of 612 nm in cyclohexane to 585 nm in ethanol at 298 K, contrary to the usual dispersion red shift. A small mechanical solvent-cage effect is noted in ethanol at 77 K, but solvent dielectric relaxation is not apparent from the fluorescence spectrum. Thus, HAP serves to dis h static solvent-cage polarity from dynamical solvent dielectric relaxation and other solvent-cage effects (mechanical restriction of molecular conformation). HAP as a PT-fluorescence probe is applied to human serum albumin (HSA) and beaver apomyoglobin.Fluorescent-probe spectroscopy of proteins as a methodology yielding structural and dynamical information concerning the chromophore (probe) environment has a long history of the application of excited-state molecular interaction between a probe and its environment (1-4). This interaction is effective for the description of both dipole-reorientational dynamics of molecules surrounding the chromophore and their dielectric properties. Nevertheless, when one is interested in structural changes in the protein as well as the intrinsic structure, such a duality can complicate the interpretation of experimental fluorescence data. Both the instantaneous electronic response of the static environment and the relaxation dynamics of the probe environment can induce analogous spectral shifts of fluorescence.In principle, several static and dynamical aspects ofprotein binding site and protein conformation could be considered. The polarity of a binding site and its dimensional and conformational features can be considered as static aspects. On the other hand, dynamical aspects could include dielectric relaxation-e.g., solvent-cage response to dipolar changes in magnitude and reorientation upon electronic excitation of a fluorescence probe-and response to conformational changes of the probe itself. The effect of the protein binding site as a "solvent cage" can then serve to separate dielectric effects from mechanical conformational changes, as deduced from electronic spectral changes.During the last decade molecular spectroscopy made great advances in the understanding of different modes of molecular excitation, such as fluorescences arising from (i) an electron-transfer excitation, (ii) proton transfer (PT), and (iii) twisted-intramolecular charge transfer. In electron transfer (ET) spectroscopy, the generation of a charge-separated polar state provokes a profound response of the environment of the fluorescence probe. In excited-state intramolecular PT (ESIPT) spectroscopy, a gross frequency...

A protein f luorescence probe system, coupling excited-state intermolecular Förster energy transfer and intramolecular proton transfer (PT), is presented. As an energy donor for this system, we used tryptophan, which transfers its excitation energy to 3-hydroxyf lavone (3-HF) as a f lavonol prototype, an acceptor exhibiting excited-state intramolecular PT. We demonstrate such a coupling in human serum albumin-3-HF complexes, excited via the single intrinsic tryptophan (Trp-214). Besides the PT tautomer f luorescence ( max ‫؍‬ 526 nm), these protein-probe complexes exhibit a 3-HF anion emission ( max ‫؍‬ 500 nm). Analysis of spectroscopic data leads to the conclusion that two binding sites are involved in the human serum albumin-3-HF interaction. The 3-HF molecule bound in the higher affinity binding site, located in the IIIA subdomain, has the association constant (k 1 ) of 7.2 ؋ 10 5 M ؊1 and predominantly exists as an anion. The lower affinity site (k 2 ‫؍‬ 2.5 ؋ 10 5 M ؊1 ), situated in the IIA subdomain, is occupied by the neutral form of 3-HF (normal tautomer). Since Trp-214 is situated in the immediate vicinity of the 3-HF normal tautomer bound in the IIA subdomain, the intermolecular energy transfer for this donor͞ acceptor pair has a 100% efficiency and is followed by the PT tautomer f luorescence. Intermolecular energy transfer from the Trp-214 to the 3-HF anion bound in the IIIA subdomain is less efficient and has the rate of 1.61 ؋ 10 8 s ؊1 , thus giving for the donor͞acceptor distance a value of 25.5 Å.Excitation by light may initiate both intramolecular and intermolecular transformations of a molecule. Among excited-state intramolecular processes, the most important are electron transfer (1-4), proton transfer (PT; refs. 5 and 6), and conformational transformation (e.g., twisting, torsion, and isomerization; refs. 7-9). The most conspicuous intermolecular events are electron, proton, and energy transfer (10, 11). Excited-state transformations can be coupled with each other and with the surrounding medium. Dependence of the excitedstate intramolecular electron transfer on environmental relaxation (2, 3) and the twisting of solute groups in response to the excited-state charge transfer (12-15) are well-established phenomena. When joined in one molecular system, excited-state events can compete dynamically. An example of such competition is the mutual exclusion of intramolecular electron and PT in 4Ј-diethylamino-3-hydroxyflavone (16-19). Proper pairing of excited-state transformations can be a source of structural͞dynamical information about the molecules possessing these transformations, the excited-state transformations themselves, and the medium where the coupling takes place.The present paper focuses on the coupling between the Förster intermolecular energy transfer (11,20,21) and intramolecular PT (refs. 6 and 22; Fig. 1). The first step of such a coupling is the R Ϫ6 -dependent (R is the intermolecular distance) dipole-dipole intermolecular energy transfer from excited donor to unexcite...