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

Photoionization and dynamic solvation of the excited states of 7-azaindole

The excited-state photophysics of the biological probe, 7-azaindole, are examined in water and methanol. Electrons in a presolvated state absorbing in the infrared appear within the excitation pulse width of 130 fs. 330 i 100 fs is required for the presolvated electron to achieve the spectrum characteristic of the completely solvated electron. An excited-state transient absorbance decays in -350 fs for 7-azaindole and its methylated analog, N1-methyl-7-azaindole (1M7AI), in the region 400-450 nm in water and methanol. The instantaneous appearance of the electron in the infrared is attributed to the decay of the lLb excited-state that overlaps the \u27La excited state of 7-azaindole. The rapid decay of the excited-state transient absorbance is attributed to preferential, dynamic solvation of the \u27La state. 7-Azaindole thus provides an interesting example of a molecule whose excited state is continuously and dynamically solvated but which also produces a species, e,,-, whose solvation appears to occur in a stepwise process

Novel noninvasive in situ probe of protein structure and dynamics

7-Amtryptophan is an ideal noninvasive in situ probe of protein structure and dynamics and provides an alternative to the use of ,tryptophan. 7-Azatryptophan affords a single-exponential fluorescence decay in aqueous solution, unlike tryptophan. Its absorption and fluorescence spectra are distinguishable from those of tryptophan. Its fluorescence spectrum and lifetime are sensitive to the environment. It can be used in peptide synthesis, and it can be incorporated into bacterial protein. These facts render 7-azatryptophan a unique probe that has the potential for widespread use

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.Reprinted (adapted) wit permission from Journal of Physical Chemistry 95 (1991): 8663, doi: 10.1021/j100175a046. Copyright 1991 American Chemical Society.</p

Photoionization and dynamic solvation of the excited states of 7-azaindole

The excited-state photophysics of the biological probe, 7-azaindole, are examined in water and methanol. Electrons in a presolvated state absorbing in the infrared appear within the excitation pulse width of 130 fs. 330 i 100 fs is required for the presolvated electron to achieve the spectrum characteristic of the completely solvated electron. An excited-state transient absorbance decays in -350 fs for 7-azaindole and its methylated analog, N1-methyl-7-azaindole (1M7AI), in the region 400-450 nm in water and methanol. The instantaneous appearance of the electron in the infrared is attributed to the decay of the lLb excited-state that overlaps the 'La excited state of 7-azaindole. The rapid decay of the excited-state transient absorbance is attributed to preferential, dynamic solvation of the 'La state. 7-Azaindole thus provides an interesting example of a molecule whose excited state is continuously and dynamically solvated but which also produces a species, e,,-, whose solvation appears to occur in a stepwise process.Reprinted (adapted) with permission from Journal of Physical Chemistry 97 (1993): 5046, doi: 10.1021/j100121a032. Copyright 1993 American Chemical Society.</p

Novel noninvasive in situ probe of protein structure and dynamics

7-Amtryptophan is an ideal noninvasive in situ probe of protein structure and dynamics and provides an alternative to the use of ,tryptophan. 7-Azatryptophan affords a single-exponential fluorescence decay in aqueous solution, unlike tryptophan. Its absorption and fluorescence spectra are distinguishable from those of tryptophan. Its fluorescence spectrum and lifetime are sensitive to the environment. It can be used in peptide synthesis, and it can be incorporated into bacterial protein. These facts render 7-azatryptophan a unique probe that has the potential for widespread use.Reprinted (adapted) with permission from Journal of the American Chemical Society 112 (1990): 7419, doi: 10.1021/ja00176a066. Copyright 1990 American Chemical Society.</p

Ultrafast ligand rebinding in the heme domain of the oxygen sensors FixL and Dos: General regulatory implications for heme-based sensors

Heme-based oxygen sensors are part of ligand-specific two-component regulatory systems, which have both a relatively low oxygen affinity and a low oxygen-binding rate. To get insight into the dynamical aspects underlying these features and the ligand specificity of the signal transduction from the heme sensor domain, we used femtosecond spectroscopy to study ligand dynamics in the heme domains of the oxygen sensors FixL from Bradyrhizobium japonicum (FixLH) and Dos from Escherichia coli (DosH). The heme coordination with different ligands and the corresponding ground-state heme spectra of FixLH are similar to myoglobin (Mb). After photodissociation, the excited-state properties and ligand-rebinding kinetics are qualitatively similar for FixLH and Mb for CO and NO as ligands. In contrast to Mb, the transient spectra of FixLH after photodissociation of ligands are distorted compared with the ground-state difference spectra, indicating differences in the heme environment with respect to the unliganded state. This distortion is particularly marked for O(2). Strikingly, heme–O(2) recombination occurs with efficiency unprecedented for heme proteins, in ≈5 ps for ≈90% of the dissociated O(2). For DosH–O(2), which shows 60% sequence similarity to FixLH, but where signal detection and transmission presumably are quite different, a similarly fast recombination was found with an even higher yield. Altogether these results indicate that in these sensors the heme pocket acts as a ligand-specific trap. The general implications for the functioning of heme-based ligand sensors are discussed in the light of recent studies on heme-based NO and CO sensors

Picosecond primary structural transition of the heme is retarded after nitric oxide binding to heme proteins

We investigated the ultrafast structural transitions of the heme induced by nitric oxide (NO) binding for several heme proteins by subpicosecond time-resolved resonance Raman and femtosecond transient absorption spectroscopy. We probed the heme iron motion by the evolution of the iron-histidine Raman band intensity after NO photolysis. Unexpectedly, we found that the heme response and iron motion do not follow the kinetics of NO rebinding. Whereas NO dissociation induces quasi-instantaneous iron motion and heme doming (< 0.6 ps), the reverse process results in a much slower picosecond movement of the iron toward the planar heme configuration after NO binding. The time constant for this primary domed-to-planar heme transition varies among proteins (∼30 ps for myoglobin and its H64V mutant, ∼15 ps for hemoglobin, ∼7 ps for dehaloperoxidase, and ∼6 ps for cytochrome c) and depends upon constraints exerted by the protein structure on the heme cofactor. This observed phenomenon constitutes the primary structural transition in heme proteins induced by NO binding