Excited-state intramolecular proton transfer as a fluorescence probe for protein binding-site static polarity.

Abstract

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 coupled proton-transfer and twisting-motion fluorescence probe for lipid bilayers

“…These changes generally are observed by monitoring the variation of photophysical properties, such as fluorescence anisotropy and lifetime, spectral position, and fluorescence quantum yield of the dye within the bilayers (1)(2)(3)(4). Recently, molecules showing excited-state protontransfer (ESPT) reactions have been suggested as probes for the study of protein conformation and binding-sites (5)(6)(7)(8). ESPT reactions are very fast elementary processes (9)(10)(11) involved in various area of physics, chemistry, and biology (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18).…”

“…Recently, molecules showing excited-state protontransfer (ESPT) reactions have been suggested as probes for the study of protein conformation and binding-sites (5)(6)(7)(8). ESPT reactions are very fast elementary processes (9)(10)(11) involved in various area of physics, chemistry, and biology (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Because of the observed large Stokes-shifted fluorescence of the proton-transfer band in these systems, such molecules might show some advantages with respect to others having a normal Stokes-shifted emission band.…”

Badania spektroskopowe efektów podwójnej fluorescencji w wybranym związku z grupy 1,3,4-tiadiazoli w rozpuszczalnikach organicznych oraz roztworach wodnych

“…Comparison with other compounds which demonstrate dual fluorescence leads to the conclusion that this phenomenon in FABT is not related to TICT, PICT or ESPIT and cannot be fully explained based on these theories [25][26][27][28][29][30][31][32][33][34]. The results obtained suggest that the dual fluorescence effect in FABT and similar thiadiazole derivatives is more likely resultant of both chromophore aggregation and molecular conformation.…”

“…Importantly, the acceptor and donor fragments of the electron have to be located in close proximity to molecules exhibiting CT (or TICT). An excited-state intramolecular proton transfer (ESIPT) is another process that allows the explanation of dual fluorescence effects [31][32][33]. This model requires a close proximity between the potential proton acceptor and the proton donor group [34].…”

Theoretical Study of Structure, Energetic, Hydrogen Bonds Strength and Intramolecular Proton Transfer of 2-(2-R (ROH, NH2, SH) Phenyl (or Pyridyl)) Benzoxazoles (or Benzothiazoles)

“…In recent years, intramolecular proton transfer compounds have attracted wide attention from scholars who specialized on the general application of these compounds in polymer stabilizers, 1 molecular energy storage devices, 2 environmental probes in bimoleculars 3 and antitumor activity. 4 Hydrogen bonds play an important role in the proton transfer reactions.…”