223 research outputs found
Photophysics of a novel optical probe: 7-azaindole
7-Azaindole is the chromophoric side chain of the nonnatural amino acid 7-azatryptopha11, which we have shown can be incorporated into bacterial protein and is amenable to peptide synthesis. Timeresolved fluorescence measurements of 7-amindole are performed as a function of solvent, pH, and temperature in order to characterize its behavior and to establish criteria for the interpretation of its photophysics when it is incorporated into, or interacts with, proteins. The first time-resolved measurements of 7-azaindole in water are presented. The dependence of the fluorescence properties of 7-azaindole in water with respect to that in various solvents of differing polarity and the temperature dependence of the fluorescence lifetimes of 7-azaindole in H20 and D20, and in CHBOH and CH30D, suggest that the fuorescent species of 7-azaindole in water is a tautomerized excited-state solutesolvent complex. Time-resolved fluorescence measurements as a function of temperature verify the existence in methanol of a ground-state precursor to the 7-azaindole *tautomer” species. Upon optical excitation, this precursor decays into the tautomer in less than 30 ps. Our results are used to rationalize the sensitivity of the fluorescence lifetime of a synthetic peptide containing 7-azatryptophan alone in aqueous solution and in complex with a protein
Synthesis and Photophysics of the Optical Probe N1-Methyl-7-azatryptophan
The development of a new intrinsic optical probe of protein structure and dynamics, Nl-methyl-7- azatryptophan (1M7AT), is reported. The utility of this nonnatural amino acid derivative lies in its single-exponential, long-lived fluorescence decay (21.7 f 0.4 ns) and in its high fluorescence quantum yield (0.53 f 0.07). Its absorption and emission maxima are red-shifted 10 and 65 nm, respectively, from those of tryptophan. These characteristics permit its unambiguous detection with unprecedented discrimination against emission from multiply occurring native tryptophan residues. In a mixture of these two amino acids, no tryptophan signal is detected until the tryptophan: N1-methyl-7-azatryptopharna tio exceeds 75: 1. Consequently, NI-methyl-7-azatryptophains ideal for studying the interactions of small peptides containing it with large proteins
Fluorescent species of 7-azaindole and 7-azatryptophan in water
A study of the fluorescence lifetimes and quantum yields of 7-azaindole and its methylated derivatives NImethyl- Famindole (1 M7AI) and 7-methyl-7H-pyrrolo[ 2,341 pyridine (7M7AI) in water is performed in order to explain the observation that the fluorescence spectrum of 7-azaindole apparently consists of one band (A, = 386 nm) whereas in alcohols the spectrum is bimodal (e.g., for methanol, A,, = 374, 505 nm). Careful measurements of the fluorescence decay as a function of emission wavelength indicate a small amplitude of an -70-ps decaying component at the bluer wavelengths and a rising component of the same duration at the redder wavelengths. The small amplitude component, which comprises no more than 20% of the fluorescence decay, is attributed to excited-state tautomerization that is mediated by the solvent. Particular attention is paid to the pH dependence of the fluorescence lifetimes and yields. We propose that upon tautomerization the basic l-nitrogen (NIo)f 7-azaindole is rapidly protonated givingrise to a species whose emission maximum is at -440 nm. The fluorescence emission maximum and lifetime of 7-azaindole is dominated by the 80% of the solute molecules that are blocked by unfavorable solvation from executing excited-state tautomerization. It is proposed that 210 ns is required for the surrounding water molecules to attain a configuration about 7-azaindole that is propitious for tautomerization
Temperature Dependence of the Excited-State Intramolecular Proton Transfer Reaction in Hypericin and Hypocrellin A
The excited-state intramolecular proton-transfer reactions of hypericin and hypocrellin A are measured as a function of temperature in an ethanol/methanol mixture. The data yield activation energies of 0.044 ± 0.008 and 2.12 ± 0.070 kcal/mol for hypericin and hypocrellin A, respectively. The negligible activation energy of hypericin is consistent with previous suggestions that the proton-transfer reaction is adiabatic (K. Das et al., J. Phys. Chem. 1997,101A, 3241.) and that a very low-amplitude displacement in at least one other coordinate be taken into account in order to describe the reaction dynamics. The proton transfer for hypocrellin is also considered to occur in the adiabatic regime, but the significant activation energy suggests that a larger amplitude motion than that for the case of hypericin comprises part of the reaction coordinate. Much of the barrier cited above for hypocrellin A results from the temperature dependence of the viscosity of the solvent mixture. The viscosity independent part of the activation barrier is 0.41 ± 0.088 kcal/mol
Probing Solvation by Alcohols and Water with 7-Azaindole
The nonradiative pathways of 7-azaindole are extremely sensitive to solvent. In alcohols, 7-azaindole executes an excited-state double-proton transfer. In water, this tautomerization is frustrated. Proton inventory experiments suggest a concerted double-proton transfer in the alcohols and point to another nonradiative process in water. We propose the following idealized picture. Whereas at room temperature 7-azaindole can form a cyclic hydrogen-bonded intermediate with a single alcohol molecule facilitating tautomerization, in water more than one solvent molecule coordinates to the solute and thus prohibits the concerted process. More detailed measurements, however, indicate that water and alcohols do not solvate 7-azaindole in fundamentally different ways, but rather that they represent two extremes of the same phenomenon
Roles of Oxygen and Photoinduced Acidification in the Light-Dependent Antiviral Activity of Hypocrellin A
Hypocrellin A displays photoinduced antiviral activity, in particular against the human immunodeficiency virus (HIV), as does its counterpart, hypericin. Although hypocrellin A, like hypericin, executes an excited-state intramolecular proton transfer, it differs from hypericin in two important ways. Unlike hypericin, hypocrellin A absolutely requires oxygen for its antiviral activity. Also, whereas we have previously demonstrated that hypericin functions as a light-induced proton source, we do not observe that hypocrellin A acidifies its surrounding medium in the presence of light. These results are discussed in the context of the ground- and excited-state photophysics of hypericin and its mechanisms of photoinduced virucidal activity
Assessing the Roles of the Constituents of Ionic Liquids in Dynamic Solvation: Comparison of an Ionic Liquid in Micellar and Bulk Form
Dynamic solvation of the dye, coumarin 153, is compared in an ionic liquid that forms micelles in water against the bulk solvent. This provides the unprecedented opportunity of investigating the behavior of the ionic liquid in two globally different configurations. It is proposed that the imidazolium moiety is in both cases responsible for the majority of the solvation, which manifests itself in the first 100 ps. Exploiting the use of ionic liquids capable of accommodating specific structures thus provides a deeper insight into how solutes interact with these fascinating and interesting solvents (at least those that are imidazolium based) that are gaining ever increasing interest in the scientific community
Using 7-Azatryptophan To Probe Small Molecule-Protein Interactions on the Picosecond Time Scale: The Complex of Avidin and Biotinylated 7-Azatryptophan
The utility of 7-azatryptophan as an alternative to tryptophan for optically probing protein structure and dynamics is demonstrated by investigating the complex of egg-white avidin and biotinylated 7-azatryptophan. We report the synthesis of biotinylated 7-azatryptophan and optical measurements of its complex with avidin. Although there are four biotin binding sites, the emission from the 7-azatryptophan tagged to biotin decays by a single exponential, whereas the tryptophyl emission from avidin requires two exponentials in order to be adequately fit. Fluorescence depolarization measurements of the complex probed by emission from 7-azatryptophan reveal both rapid (-80 ps) and much longer-lived decay. The former component is attributable to the local motion of the probe with respect to the protein; the latter component represents overall protein tumbling. In addition, energy transfer from tryptophan to 7-azatryptophan and a blue-shift in the spectrum of biotinylated 7-azatryptophan are observed upon formation of the complex. Modified strategies of effecting optical selectivity are also discussed
Effects of Distal Pocket Mutations on the Geminate Recombination of NO with Leghemoglobin on the Picosecond Time Scale
The picosecond NO geminate rebinding kinetics of wild-type leghemoglobin, a monomeric plant hemoglobin with structural similarity to myoglobin, and six mutant proteins at the distal histidine (H61G, H61A, H61V, H61L, H61R, H61F) are investigated. All of the mutant proteins yield rebinding kinetics that are initially more rapid than that of the wild-type protein. At long times, the rebinding of H61F becomes slower than that of wild-type leghemoglobin. The H61V, H61L, and H61G mutant proteins give extraordinarily rapid and complete geminate rebinding. On a 40 ps time scale, distal effects are overwhelmingly evident for all of the mutants considered. That binding is both rapid and, in several cases, essentially single-exponential is suggestive of the nature of the barrier induced by the distal modification: it must be such that the ligand is prohibited from reorienting with respect to, and diffusing sufficiently far from, the heme iron so that a distribution of return paths is not offered to it. Over the past 20 years, the relative importance attributed to the proximal and the distal sides in modulating geminate ligand binding has varied considerably. Our results with leghemoglobin are discussed in terms of the relative contributions of proximal and distal effects to geminate rebinding kinetics
A Comparative Femtosecond Coherence Study of the Unligated Monomeric Hemeproteins Myoglobin and Leghemoglobin
Impulsive optical excitation has been performed on wild type, unligated leghemoglobin for the first time to compare the induced vibrational coherence with that observed in myoglobin. Both proteins were excited at the Soret maxima and probed at red and blue edges of the Soret band. The resulting kinetic traces were modulated by low-frequency vibrations. Leghemoglobin shows a decrease in vibrational amplitude compared with myoglobin. The possible cause for the amplitude differences is discussed in terms of contributions from both ground- and excited-state vibrational coherences and ground-state heterogeneity
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