Interaction of fluorescent adenine nucleotide derivatives with the ADP/ATP carrier in mitochondria. 1. Comparison of various 3'-O-ester adenine nucleotide derivatives

Fluorescent 3'-O-acyl-substituted adenine nucleotide (dimethylamino)naphthoyl and trinitrophenyl groups were studied for binding to the ADP/ATP carrier in mitochondria and submitochondrial particles. The changes in fluorescence intensity and emission maximum are for the most part similar to those observed in nonaqueous solvents. The (dimethylamino)naphthoyl derivatives from a largely quenched aqueous state have a shortwave shift up to 85 nm and increase up to 90-fold (1,5 derivative), whereas the little quenched naphthoyl derivatives show a fluorescence decrease and the weakly fluorescent trinitrophenyl derivative shows only a small increase on binding. All derivatives are good inhibitors (K1 = 1-10 microM) of nucleotide transport. The fluorescence titrations have an apparent K1/2 = 2-7 microM. The fluorescence of the 1,5-DAN nucleotide is fully suppressed by bongkrekate but only partially suppressed by carboxyatractylate. The fluorescence response is much stronger in submitochondrial particles than in mitochondria. Both facts suggest fluorescent binding to the "m" state of the carrier site at the inner face of the membrane. 1,5-DAN-AMP shows a slightly more efficient binding than DAN-ADP or DAN-ATP.     

Picosecond-nanosecond laser photolysis studies of a photoacid generator in solutions: Transient absorption spectroscopy and transient grating measurements.

Photochemical reaction profiles of a photoacid generator, N-trifluoromethylsulfonyloxy-1,8-naphthalimide (NI-Sf), in solution phase were investigated by means of picosecond and nanosecond transient absorption spectroscopy as well as transient grating measurements. Picosecond transient absorption spectroscopy directly revealed that the lifetime of the S1 state and the intersystem-crossing yield decreased with increasing solvent polarity. On the other hand, photochemical reaction yield increased with an increase in the solvent polarity. These results indicated that the photochemical reaction started in the S1 state. Transient grating measurement directly detected the diffusion process of the proton and its diffusion coefficient was obtained to be 3.9 x 10(-9) m2 s(-1), which was several times larger than those of the usual solute molecules.     

Photophysics of the fluorescent K+ indicator PBFI.

The fluorescent indicator PBFI is widely used for the determination of intracellular concentrations of K+. To investigate the binding reaction of K+ to PBFI in the ground and excited states, steady-state and time-resolved measurements were performed. The fluorescence decay surface was analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution at pH 7.2: k01 = 1.1 x 10(9) s-1, k21 = 2.7 x 10(8) M-1s-1, k02 = 1.8 x 10(9) s-1, and k12 = 1.4 x 10(9) s-1. k01 and k02 denote the respective deactivation rate constants of the K+ free and bound forms of PBFI in the excited state. k21 represents the second-order rate constant of binding of K+ to the indicator in the excited state whereas k12 is the first-order rate constant of dissociation of the excited K(+)-PBFI complex. From the estimated values of k12 and k21, the dissociation constant Kd* in the excited state was calculated. It was found that pKd* (-0.7) is smaller than pKd (2.2). The effect of the excited-state reaction can be neglected in the determination of Kd and/or the K+ concentration. Therefore, intracellular K+ concentrations can be accurately determined from fluorimetric measurements by using PBFI as K+ indicator.     

Determination of rates and yields of interchromophore (folate----flavin) energy transfer and intermolecular (flavin----DNA) electron transfer in Escherichia coli photolyase by time-resolved fluorescence and absorption spectroscopy

Escherichia coli DNA photolyase, which photorepairs cyclobutane pyrimidine dimers, contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and 5,10-methenyltetrahydrofolate (MTHF). Previous work has shown that MTHF is the primary photoreceptor which transfers energy to the FADH2 cofactor; the FADH2 singlet excited state then repairs the photodimer by electron transfer. In this study, we have determined the rate constants for these photophysical processes by time-resolved fluorescence and absorption spectroscopy. From time-resolved fluorescence, we find that energy transfer from MTHF to FADH2 and FADH degrees occurs at rates of 4.6 x 10(9) and 3.0 x 10(10) s-1, respectively, and electron transfer from FADH2 to a pyrimidine dimer occurs at a rate of 5.5 x 10(9) s-1. Using F?rster theory for long-range energy transfer and assuming K2 = 2/3, the interchromophore distances were estimated to be 22 A in the case of the MTHF-FADH2 pair and 21 A for the MTHF-FADH degrees pair. Picosecond absorption spectroscopy identified an MTHF single state which decays to yield the first excited singlet state of FADH2. The lifetimes of MTHF and FADH2 singlets and the rates of interchromophore energy transfer, as well as the rate of electron transfer from FADH2 to DNA measured by time-resolved fluorescence, were in excellent agreement with the values obtained by picosecond laser flash photolysis. Similarly, fluorescence or absorption lifetime studies of the folate-depleted enzyme with and without photodimer suggest that FADH2, in its singlet excited state, transfers an electron to the dimer with 89% efficiency. The distance between FADH2 and the photodimer was calculated to be ca. 14 A.     

Photophysics of the fluorescent Ca2+ indicator Fura-2.

The photophysics of the complex forming reaction of Ca2+ and Fura-2 are investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with BAPTA as Ca2+ buffer: k01 = 1.2 x 10(9)s-1, k21 = 1.0 x 10(11) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 2.2 x 10(7) s-1, and with EGTA as Ca2+ buffer: k01 = 1.4 x 10(9) s-1, k21 = 5.0 x 10(10) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 3.2 x 10(7) s-1. k01 and k02 denote the respective deactivation rate constants of the Ca2+ free and bound forms of Fura-2 in the excited state. k21 represents the second-order rate constant of binding of Ca2+ and Fura-2 in the excited state, whereas k12 is the first-order rate constant of dissociation of the excited Ca2+:Fura-2 complex. The ionic strength of the solution was shown not to influence the recovered values of the rate constants. From the estimated values of k12 and k21, the dissociation constant K*d in the excited state was calculated. It was found that in EGTA Ca2+ buffer pK*d (3.2) is smaller than pKd (6.9) and that there is negligible interference of the excited-state reaction with the determination of Kd and [Ca2+] from fluorimetric titration curves. Hence, Fura-2 can be safely used as an Ca2+ indicator. From the obtained fluorescence decay parameters and the steady-state excitation spectra, the species-associated excitation spectra of the Ca2+ free and bound forms of Fura-2 were calculated at intermediate Ca2+ concentrations.     

One- and two-photon-induced fluorescence from recombinant green fluorescent protein

The fluorescence spectral properties of recombinant green fluorescent protein (rGFP) were examined with one- and two-photon excitations using femtosecond pulses from a Ti:sapphire laser. Intensity-dependent properties of the two-photon-induced fluorescence from rGFP excited by an 800-nm, 100-fs laser beam were reported, and the two-photon excitation cross section of rGFP was measured at 800 nm as about 160 x 10(-50) cm(4)s/photon. The possible excited-state proton transfer between two electronic states at about 400 nm in protonated (RH) species and 478 nm in deprotonated (R(-)) species in rGFP was confirmed by fluorescence and fluorescence excitation anisotropy spectra. A subelectronic state (or vibronic progression) at about 420 nm in RH species was identified, which was relatively stable and not involved in the excited state proton transfer in rGFP upon irradiation.     

Intramolecular and intermolecular hydrogen-bonding effects on photophysical properties of 2'-aminoacetophenone and its derivatives in solution.

Effects of intra- and intermolecular hydrogen-bonds on the photophysical properties of 2'-aminoacetophenone derivatives (X-C6H4-COCH3) having a substituted amino group (X) with different hydrogen-bonding ability to the carbonyl oxygen (X: NH2(AAP), NHCH3(MAAP), N(CH3)2(DMAAP), NHCOCH3(AAAP), NHCOCF3(TFAAP)) are investigated by means of steady-state and time-resolved fluorescence spectroscopy and time-resolved thermal lensing. Based on the photophysical parameters obtained in aprotic solvents with different polarity and protic solvents with different hydrogen-bonding ability, the characteristic photophysical behavior of the 2'-aminoacetophenone derivatives is discussed in terms of hydrogen-bonding and n,pi*-pi,pi* vibronic coupling. The dominant deactivation process of AAP and MAAP in nonpolar aprotic solvents is the extremely fast internal conversion (k(ic)= 1.0 x 10(11) s(-1) for AAP and 3.9 x 10(10) s(-1) for MAAP in n-hexane). The internal conversion rates of both compounds decrease markedly with increasing solvent polarity, suggesting that vibronic interactions between close-lying S1(pi,pi*) and S2(n,pi*) states lead to the large increase in the non-radiative decay rate of the lowest excited singlet state. It is also suggested that for MAAP, which has a stronger hydrogen-bond as compared to AAP, an intramolecular hydrogen-bonding induced deactivation is involved in the dissipation of the S1 state. For DMAAP, which cannot possess an intramolecular hydrogen-bond, the primary relaxation mechanism of the S1 state in nonpolar aprotic solvents is the intersystem crossing to the triplet state, whereas in protic solvents very efficient internal conversion due to intermolecular hydrogen-bonding is induced. In contrast, the fluorescence spectra of AAAP and TFAAP, which have an amino group with a much stronger hydrogen-bonding ability, give strongly Stokes-shifted fluorescence, indicating that these compounds undergo excited-state intramolecular proton transfer reaction upon electronic excitation.     

New method for characterizing highly disulfide-bridged peptides in complex mixtures: application to toxin identification from crude venoms

Animal venoms are highly complex mixtures that can contain many disulfide-bridged toxins. This work presents an LC-MALDI approach allowing (1) a rapid classification of toxins according to their number of disulfide bonds and (2) a rapid top-down sequencing of the toxins using a new MALDI matrix enhancing in-source decay (ISD). The crude venom is separated twice by LC: the fractions of the first separation are spotted on the MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) and the others using 1,5-diaminonaphthalene (1,5-DAN). CHCA spots are more convenient for obtaining a precise mass fingerprint of a large number of peptides; however, the analysis of 1,5-DAN spots allows the number of disulfide bridges to be counted owing to their partial in-plume reduction by this particular matrix. Subsequently, the disulfide bonds of all peptides present in the crude venom were reduced by an excess of tris(carboxyethyl)phosphine before the LC separation and were subjected to the same analysis in CHCA and 1,5-DAN. Toxins were sequenced using a TOF/TOF analysis of metastable fragments from CHCA spots and ISD fragmentation from 1,5-DAN spots. Novel conotoxin sequences were found using this approach. The use of 1,5-DAN for ISD top-down sequencing is also illustrated for higher molecular weight toxins such as snake cardiotoxins and neurotoxins (>6500 Da), where sequence coverage >70% is obtained from the c-ion series.     

Preliminary studies of the enhanced electrochemiluminescence of 2,3-diaminonaphthalene in the presence of specific metal ions

Electrochemiluminescence (ECL) of 200 ppm 2,3-diaminonaphthalene (2,3-DAN) was studied alone and in conjunction with 100 ppm of 34 different metal and non-metal ions and revealed three relatively intense ECL responses from interactions of 2,3-DAN with Au⁺, Fe⁺³ and V⁺⁵. ECL responses from Cr⁺⁶ or Ru⁺³ with 2,3-DAN were less intense, but noteworthy, as was the coloured fluorescent product of the non-metal ion Se⁺⁴ interaction with 2,3-DAN. Several intense 2,3-DAN–metal ion ECL reactions were studied in greater detail and revealed various titration curves with ionic detection limits in the low ppm range, using a fixed level (200 ppm) of 2,3-DAN. © 1998 John Wiley & Sons, Ltd.     

Protonation of excited state pyrene-1-carboxylate by phosphate and organic acids in aqueous solution studied by fluorescence spectroscopy

Pyrene-1-carboxylic acid has a pK of 4.0 in the ground state and 8.1 in the singlet electronic excited state. In the pH range of physiological interest (pH approximately 5-8), the ground state compound is largely ionized as pyrene-1-carboxylate, but protonation of the excited state molecule occurs when a proton donor reacts with the carboxylate during the excited state lifetime of the fluorophore. Both forms of the pyrene derivatives are fluorescent, and in this work the protonation reaction was measured by monitoring steady-state and time-resolved fluorescence. The rate of protonation of pyrene-COO(-) by acetic, chloroacetic, lactic, and cacodylic acids is a function of DeltapK, as predicted by Marcus theory. The rate of proton transfer from these acids saturates at high concentration, as expected for the existence of an encounter complex. Trihydrogen-phosphate is a much better proton donor than dihydrogen- and monohydrogen-phosphate, as can be seen by the pH dependence. The proton-donating ability of phosphate does not saturate at high concentrations, but increases with increasing phosphate concentration. We suggest that enhanced rate of proton transfer at high phosphate concentrations may be due to the dual proton donating and accepting nature of phosphate, in analogy to the Grotthuss mechanism for proton transfer in water. It is suggested that in molecular structures containing multiple phosphates, such as membrane surfaces and DNA, proton transfer rates will be enhanced by this mechanism.     

Involvement of Hydroxyl Radical Formation and Dna Strand Breakage in the Cytotoxicity of Anthraquinone Antitumour Agents

Four 9,10-anthraquinones (AQ) mono- or bis-substituted with the -NH(CH₂)₂ NH(CH₂)₂OH group were studied. 1-AQ, 1,5-AQ and 1,8-AQ but not 1,4-AQ (100°M) generated pBR322 plasmid DNA single strand breaks in the presence of purified NADPH dependent cytochrome P450 reductase. 1-AQ, 1,5-AQ and 1,8-AQ (at 100 °M) stimulated hydroxyl radical formation in MCF-7 S9 cell fraction (as measured by dimethyl pyrolline N-oxide spin trapping) and MCF-7 DNA strand breaks as measured by alkaline filter elution. In contrast 1,4-AQ did not stimulate hydroxyl radical formation and produced considerably less strand breaks in MCF-7 cells compared to the other AQ's. It would appear that the position of the -NH(CH₂)₂ NH(CH₂)₂OH groups on the chromophore is an important determinant in the metabolic activation of cytotoxic anthraquinones. This may contribute to the cytotoxicity (ID₅₀ values) of 1-AQ (0.06 °M), 1-8-AQ (0.5 °M) and 1,5-AQ (12.3 °M) but not the 1,4-AQ (1.2 °M).     

Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes.

In this work we have applied a kinetic scheme derived from fluorescence kinetics of pyrene-labeled phosphatidylcholine in phosphatidylcholine membrane to explain the fluorescence quenching of 1-palmitoyl-2-(10-[pyrenl-yl]-sn-glycerol-3-phosphatidylchol ine (PPDPC) liposomes by tetracyanoquinodimethane (TCNQ). The scheme was also found to be applicable to neat PPDPC and the effect of the quencher could be attributed to certain steps of the proposed mechanism. The TCNQ molecules influence the fluorescence of pyrene moieties in PPDPC liposome in two ways. Firstly, an interaction between the quencher molecule and the pyrene monomer in the excited state quenches monomer fluorescence and effectively prevents the diffusional formation of the excimer. Secondly, an interaction between the quencher molecule and the excited dimer quenches the excimer fluorescence. The TCNQ molecule does not prevent the formation of the excimer in pyrene moieties aggregated in such a way that they require only a small rotational motion to attain excimer configuration. The diffusional quenching rate constant is calculated to be 1.0 x 10(8) M-1 s-1 for the pyrene monomer quenching and 1.3 x 10(7) M-1 s-1 for the pyrene excimer quenching. The diffusion constant of TCNQ is 1.5 x 10(-7) cm2 s-1 for the interaction radii of 0.8-0.9 nm. The TCNQ molecules are practically totally partitioned in the membrane phase.     

Primary steps of the photochemical reactions of 2-cyano-10-(3-[dimethylamino, N-oxide]-2-methylpropyl)-5-oxide-phenothiazine, the photoproduct of cyamemazine, a phototoxic neuroleptic: comparison with the sulfoxide.

The UVA-absorbing photoproduct resulting from the oxidation of the sulfur atom and of the side chain nitrogen of the phototoxic drug cyamemazine (CMZ) (2-cyano-10-(3-[dimethylamino]-2 methylpropyl)-phenothiazine) is a potent photodynamic photosensitizer. The photophysical and photochemical properties of this photoproduct (P) (2-cyano-10-(3-[dimethylamino, N-oxide]-2-methylpropyl)-5-oxide-phenothiazine)) have been investigated in neutral buffered aqueous solutions and in ethanol and compared to those of the sulfoxide (S) (2-cyano-10-(3-[dimethylamino]-2 methylpropyl)-5-oxide-phenothiazine), a CMZ oxidation product of cells. The fluorescence quantum yield (PhiF) of P is 0.25 and 0.21 in pH 7 phosphate buffer and ethanol, respectively. By contrast, S (PhiF = 0.14 in buffer) is practically unfluorescent in alcohol. In buffer, the fluorescence lifetimes of P and S are 10.5 and 11.8 ns, respectively. The transient absorbance of the first excited triplet state (3P1) with a characteristic absorption band peaking at 660 nm (epsilon = 5,300 M(-1) cm(-1)) has been observed by 355 nm laser flash spectroscopy of deaerated phosphate buffer or ethanol solutions. In buffer, the 3P1 lifetime is 0.5 micros. The energy transfer which occurs from the 3P1 to naproxen suggests that the 3P1 energy is greater than 62 kcal mol(-1). Triplet quenching by dioxygen occurs at rate 2.3 x 10(9) M(-1) s(-1). With the triplet benzophenone as actinometer, the 3P1 formation quantum yield is found to be 0. 40 in buffer. The 3P1 state is quenched by ethanol and 2-propanol with bimolecular reaction rate constants of 1.6 and 2.4 x 10(6) M(-1) s(-1), respectively. In buffer, P and S triplet states react with tryptophan, indole and cysteine at rate constants of the order of 10(9) M(-1) s(-1) for Trp and indole and 10(8) M(-1) s(-1) for Cys.     

Involvement of Hydroxyl Radical Formation and Dna Strand Breakage in the Cytotoxicity of Anthraquinone Antitumour Agents

Four 9,10-anthraquinones (AQ) mono- or bis-substituted with the -NH(CH₂)₂ NH(CH₂)₂OH group were studied. 1-AQ, 1,5-AQ and 1,8-AQ but not 1,4-AQ (100°M) generated pBR322 plasmid DNA single strand breaks in the presence of purified NADPH dependent cytochrome P450 reductase. 1-AQ, 1,5-AQ and 1,8-AQ (at 100 °M) stimulated hydroxyl radical formation in MCF-7 S9 cell fraction (as measured by dimethyl pyrolline N-oxide spin trapping) and MCF-7 DNA strand breaks as measured by alkaline filter elution. In contrast 1,4-AQ did not stimulate hydroxyl radical formation and produced considerably less strand breaks in MCF-7 cells compared to the other AQ's. It would appear that the position of the -NH(CH₂)₂ NH(CH₂)₂OH groups on the chromophore is an important determinant in the metabolic activation of cytotoxic anthraquinones. This may contribute to the cytotoxicity (ID₅₀ values) of 1-AQ (0.06 °M), 1-8-AQ (0.5 °M) and 1,5-AQ (12.3 °M) but not the 1,4-AQ (1.2 °M).     

Spectrofluorimetric determination of dopamine using 1,5-bis(4,6-dicholro-1,3,5-triazinylamino) naphthalene.

A new fluorescent reagent, 1,5-bis(4,6-dichloro-1,3,5-triazinylamino)naphthalene, containing two active chlorines, was synthesized by a one-step reaction. Under the optimum conditions for the determination of dopamine, the enhanced fluorescence intensity is proportional to the dopamine concentration. The fluorescence intensity was measured at lambda(ex/em) = 400/460 nm, with and without dopamine. The linear range and detection limit for the determination of dopamine were 1.0 x 10(-7) mol/L-5.0 x 10(-5) mol/L and 4.0 x 10(-8) mol/L. This method is simple, practical, can afford good precision and accuracy and can be successfully applied to assess dopamine in injections and human serum samples.     

Primary photoprocesses of phytochrome. Picosecond fluorescence kinetics of oat and pea phytochromes

The primary photoprocesses of etiolated oat and pea phytochromes (Pr forms) are diffusion-modulated by the microscopic viscosity within the chromophore pocket. The chromophore pocket is preferentially accessible to glycerol but not to Ficoll. Glycerol preferentially retarded the rate (rate constant ca. 1-2 X 10(10) s-1) of the initial reaction from the Qy excited state of phytochrome, whereas it increased the long fluorescence lifetime (nanosecond) component that can be attributed to either an emitting intermediate or to modified/conformationally heterogeneous phytochrome populations. The picosecond time-resolved fluorescence spectra of different phytochrome preparations (i.e., full-length vs 6/10-kDa NH2-terminus truncated forms of phytochromes from monocot and dicot plants) revealed no significant differences. The spectra in the picosecond time scale showed no spectral shifts, but at longer time scales of up to approximately 1.90 ns, significant blue spectral shifts were observed. The shifts were more in the truncated than in the full-length pea phytochrome. Comparison of the fluorescence decay data and the picosecond time-resolved fluorescence spectra suggests differences in conformational flexibility/heterogeneity among the preparations of the monocot vs dicot phytochromes and the full-length native vs the amino terminus truncated phytochromes.     

Laurdan solvatochromism: solvent dielectric relaxation and intramolecular excited-state reaction.

Absorption, steady-state, and time-resolved fluorescence measurements have been performed on laurdan dissolved either in white viscous apolar solvents or in ethanol as a function of temperature. The heterogeneity of the absorption spectra in white oils or in ethanol is consistent with semiempirical calculations performed previously on Prodan. From steady-state and time-resolved fluorescence measurements in apolar media, an excited state reaction is evidenced. The bimodal lifetime distribution determined from the maximum entropy method (MEM) analysis is attributed to the radiative deexcitation of a "locally excited" (LE) state and of a "charge transfer" (CT) state, whereas a very short component (20 ps), the sign and the amplitude of which depend on the emission wavelength, is attributed to the kinetics of the interconvertion reaction. The observation of an isoemissive point in the temperature range from -50 degrees C to -110 degrees C in ethanol suggests an interconvertion between two average excited-state populations: unrelaxed and solvent-relaxed CT states. A further decrease in temperature (-190 degrees C), leading to frozen ethanol, induces an additional and important blue shift. This low temperature spectrum is partly attributed to the radiative deexcitation of the LE state. Time-resolved emission spectra (TRES) measurements at -80 degrees C in the ethanol liquid phase show a large spectral shift of approximately 2500 cm(-1) (stabilization energy of the excited state: 7.1 kcal x M(-1)). The time-dependent fluorescence shift (TDFS) is described for its major part by a nanosecond time constant. The initial part of the spectral shift reveals, however, a subnanosecond process that can be due to fast internal solvent reorientation and/or to intramolecular excited-state reactions. These two relaxation times are also detected in the analysis of the fluorescence decays in the middle range of emission energy. The activation energy of the longest process is approximately 3 kcal x M(-1). At -190 degrees C, one subnanosecond and one nanosecond excited-state reactions are also evidenced. They are likely due to intramolecular rearrangements after the excitation, leading to the CT state and not to solvent relaxation, which is severely hindered in these temperature conditions. Therefore, both intramolecular and solvent relaxations are responsible for the large Stokes shift displayed by this probe as a function of solvent polarity. A possible scheme is proposed for the deexcitation pathway, taking into account the kinetics observed in these different solvents.     

First steps of retinal photoisomerization in proteorhodopsin

The early steps (<1 ns) in the photocycle of the detergent solubilized proton pump proteorhodopsin are analyzed by ultrafast spectroscopic techniques. A comparison to the first primary events in reconstituted proteorhodopsin as well as to the well known archaeal proton pump bacteriorhodopsin is given. A dynamic Stokes shift observed in fs-time-resolved fluorescence experiments allows a direct observation of early motions on the excited state potential energy surface. The initial dynamics is dominated by sequentially emerging stretching (<150 fs) and torsional (approximately 300 fs) modes of the retinal. The different protonation states of the primary proton acceptor Asp-97 drastically affect the reaction rate and the overall quantum efficiencies of the isomerization reactions, mainly evidenced for time scales above 1 ps. However, no major influence on the fast time scales (approximately 150 fs) could be seen, indicating that the movement out of the Franck-Condon region is fairly robust to electrostatic changes in the retinal binding pocket. Based on fs-time-resolved absorption and fluorescence spectra, ground and exited state contributions can be disentangled and allow to construct a reaction model that consistently explains pH-dependent effects in solubilized and reconstituted proteorhodopsin.     

Alteration of A.T base-pair opening kinetics by the ammonium cation in DNA A-tracts

We have investigated by NMR the effects of NH(4)(+) on the chemical shifts, on the structure, and on the imino proton exchange kinetics of two duplexes containing an A-tract, [d(CGCGAATTCGCG)](2) and [d(GCA(4)T(4)GC)](2), and of a B-DNA duplex,[d(CGCGATCGCG)](2). Upon NH(4)(+) addition to [d(CGCGAATTCGCG)](2), the adenosine H2 protons, the thymidine imino protons, and the guanosine imino proton of the adjacent G.C pair show unambiguous chemical shifts. Similar shifts are observed in the A-tract of [d(GCA(4)T(4)GC)](2) and for the A5(H2) proton of the B DNA duplex [d(CGCGATCGCG)](2). The localization of the shifted protons suggests an effect related to NH(4)(+) binding in the minor groove. The cross-peak intensities of the NOESY spectra collected at low and high NH(4)(+) concentrations are comparable, and the COSY spectra do not show any change of the sugar pucker. This indicates a modest effect of ammonium binding on the duplex structures. Nevertheless, the imino proton exchange catalysis by ammonia provides evidence for a substantial effect of NH(4)(+) binding on the A.T base-pair kinetics in the A-tracts. Proton exchange experiments performed at high and low NH(4)(+) concentrations show the occurrence of two native conformations in proportions depending on the NH(4)(+) concentration. The base-pair lifetimes and the open-state lifetimes of each conformation are distinct. Exchange from each conformation proceeds via a single open state. But if, and only if, the NH(4)(+) concentration is kept larger than 1 M, the A.T imino proton exchange times of A-tract sequences exhibit a linear dependence versus the inverse of the NH(3) proton acceptor concentration. This had been interpreted as an indication for two distinct base-pair opening modes (W?rml?nder, S., Sen, A., and Leijon, M. (2000) Biochemistry 39, 607-615).