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Picosecond time-resolved fluorescence spectroscopy has been applied to the studies of excited-state intramolecular proton transfer (ESIPT) dynamics in two 4‘-(dialkylamino)-3-hydroxyflavone derivatives (unsubstituted and substituted at the 6-position) in ethyl acetate and dichloromethane. In all the studied cases, the fluorescence decay kinetics of both short-wavelength normal (N*) and long-wavelength tautomer (T*) bands can be characterized by the same two lifetime components, which are constant over the all wavelength range of the emission. In the meantime, the preexponential factor of the short-lifetime component changes its sign, being positive for the N* and negative for the T* emission band. Moreover, the two preexponential factors of the T* emission decay are the same in magnitude but opposite in sign. These features are characteristic of a fast reversible two-state ESIPT reaction. Reconstruction of time-resolved spectra allows observing the evolution of these spectra with the appearance, rapid growth, and stabilization (in less than 200 ps) of the relative intensities of the two emission bands. A detailed kinetic model was applied for the analysis of these data, which involved the determination of radiative and nonradiative decay rate constants of both N* and T* forms and of forward and reverse rate constants for transitions between them. We show that ESIPT reaction in the studied conditions occurs on the scale of tens of picoseconds and thus is uncoupled with dielectric relaxations in the solvent occurring at subpicosecond times. Moreover, the radiative and nonradiative deactivation processes were found to be much slower than the ESIPT reaction, suggesting that the relative intensities of the two emission bands are mainly governed by the ESIPT equilibrium. Therefore, both electrochromic and solvatochromic effects on the relative intensities of the two emission bands in 4‘-(dialkylamino)-3-hydroxyflavones result from the shifts in the ESIPT equilibrium.

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The spectroscopic behavior of the 4′-dialkylamino-3-hydroxyflavones in protic environments is very unusual. Previous studies showed that in contrast to other solvatochromic dyes containing carbonyl group (coumarins, Nile Red, PRODAN, etc.), their Stokes shift does not increase on the formation of intermolecular H-bonds with protic solvents. The present steady-state and time-resolved studies show that the ground-state equilibrium between the H-bonded and non-H-bonded forms of this derivative in mixed solvents is not changed significantly when the dye is excited to the normal (N*) excited state. New H-bonds do not form, but those already existing in the ground state can disrupt on a slow time scale. This last process is probably coupled with the slow excited-state intramolecular proton transfer (ESIPT) reaction of the H-bonded form of the dye. Therefore, the fluorescence spectra of the dye provide a measure of the ground state distribution between its H-bonded and non-H-bonded forms, which in turn reflects the H-bonding potential of the environment. Due to this feature, this dye can serve not only as a calibrator of solvent properties but also as a unique sensor of H-bonding potential in unknown media. This sensing can be provided by the relative intensities of the two separated emission bands in the fluorescence spectra.

The nucleocapsid protein (NC) plays an important role in HIV-1, mainly through interactions with the genomic RNA and its DNA copies. Though the structures of several complexes of NC with oligonucleotides (ODNs) are known, detailed information on the ODN dynamics in the complexes is missing. To address this, we investigated the steady state and time-resolved fluorescence properties of 2-aminopurine (2Ap), a fluorescent adenine analog introduced at positions 2 and 5 of AACGCC and AATGCC sequences. In the absence of NC, 2Ap fluorescence was strongly quenched in the flexible ODNs, mainly through picosecond to nanosecond dynamic quenching by its neighboring bases. NC strongly restricted the ODN flexibility and 2Ap local mobility, impeding the collisions of 2Ap with its neighbors and thus, reducing its dynamic quenching. Phe16→Ala and Trp37→Leu mutations largely decreased the ability of NC to affect the local dynamics of 2Ap at positions 2 and 5, respectively, while a fingerless NC was totally ineffective. The restriction of 2Ap local mobility was thus associated with the NC hydrophobic platform at the top of the folded fingers. Since this platform supports the NC chaperone properties, the restriction of the local mobility of the bases is likely a mechanistic component of these properties.

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The steady-state and time-resolved fluorescence properties of two zinc-saturated 18-residue synthetic peptides with the amino acid sequence of the NH2-terminal (NCp7 13-30 F16W, where the naturally occurring Phe was replaced by a Trp residue) and the COOH-terminal (NCp7 34-51) zinc finger domains of human immunodeficiency virus type I nucleocapsid protein were investigated. Fluorescence intensity decay of both Trp 16 and Trp 37 residues suggested the existence of two fully solvent-exposed ground-state classes governed by a C = 2.2 equilibrium constant. The lifetimes of Trp 16 classes differed from those of Trp 37 essentially because of differences in nonradiative rate constants. Arrhenius plots of the temperature-dependent nonradiative rate constants suggested that the fluorescence quenchers involved in both classes and in both peptides were different and the collisional rate of these quenchers with the indole ring was very low, probably because of the highly constrained peptide chain conformation. The nature of the ground-state classes was discussed in relation to 1H nuclear magnetic resonance data. Using Trp fluorescence to monitor the interaction of both peptides with tRNA(Phe) we found that a stacking between the indole ring of both Trp residues and the bases of tRNA(Phe) occurred. This stacking constituted the main driving force of the interaction and modified the tRNA(Phe) conformation. Moreover, the binding of both fingers to tRNA(Phe) was noncooperative with similar site size (3 nucleotide residues/peptide), but the affinity of the NH2-terminal finger domain (K = 1.3 (+/- 0.2) 10(5) M-1) in low ionic strength buffer was one order of magnitude larger than the COOH-terminal one due to additional electrostatic interactions involving Lys 14 and/or Arg 29 residues.