Sensitization of lanthanides by nonnatural amino acids

The sensitization of Eu(III) and Tb(III) by ethylenediaminetetraaceticacid (EDTA)-derivatized tryptophan (Trp), 7-azatryptophan (7AW) and 5-hydroxytryptophan (5HW) has been examined. These Trp analogs were utilized in the present study because they can be incorporated into proteins in place of native Trp residues and because they absorb strongly beyond 305 nm (where Trp absorbance goes to zero), allowing selective excitation of such species in the presence of other Trp-containing proteins. All three indole derivatives were able to sensitize Tb(III) luminescence, with the relative sensitization being in the order Trp > 5HW > 7AW. On the other hand, only the 7AW-EDTA complex was able to sensitize Eu(III) luminescence, likely owing to a better spectral overlap between 7AW emission and Eu(III) absorbance. The sensitized emission of Tb(III) and Eu(II) displayed the expected long emission lifetimes at 545 nm [for Tb(III)] and 617 nm [for Eu(III)], indicating that long-lifetime lanthanide emission could be produced using nonnatural amino-acid donors. Thus, 7AW- and 5HW-sensitized lanthanide emissions should prove to be useful in biophysical studies, such as the use of fluorescence energy transfer to probe biomolecular interactions in vivo.     

On spectral relaxation in proteins

During the past several years there has been debate about the origins of nonexponential intensity decays of intrinsic tryptophan (trp) fluorescence of proteins, especially for single tryptophan proteins (STP). In this review we summarize the data from diverse sources suggesting that time-dependent spectral relaxation is a ubiquitous feature of protein fluorescence. For most proteins, the observations from numerous laboratories have shown that for trp residues in proteins (1) the mean decay times increase with increasing observation wavelength; (2) decay associated spectra generally show longer decay times for the longer wavelength components; and (3) collisional quenching of proteins usually results in emission spectral shifts to shorter wavelengths. Additional evidence for spectral relaxation comes from the time-resolved emission spectra that usually shows time-dependent shifts to longer wavelengths. These overall observations are consistent with spectral relaxation in proteins occurring on a subnanosecond timescale. These results suggest that spectral relaxation is a significant if not dominant source of nonexponential decay in STP, and should be considered in any interpretation of nonexponential decay of intrinsic protein fluorescence.     

Nonexponential Fluorescence Decay of 7-Azatryptophan Induced in a Peptide Environment

7-Azatryptophan is an alternative to tryptophan as an optical probe of protein structure and dynamics. 7-Azatryptophan is synthetically incorporated into an octapeptide (NAc-Lys-Ala-Cys-Pro-7-azatryptophan-Asn-Cys-Asp-NH₂) that mimics the active site of potato chymotrypsin inhibitor II, which is known to be a strong inhibitor of α-chymotrypsin. The synthetic octapeptide retains some of this inhibitory activity. This is the first compound containing the 7-azaindole chromophore to display a nonexponential fluorescence decay (well fit to two exponentials) in water when fluorescence is collected over the entire emission band. The effect of external quenchers on the fluorescence decay is monitored and seen to differ markedly for the two components. These results are discussed in terms of the solvation of the 7-azaindole chromophore itself, which promotes or impedes excited-state tautomerization. The fluorescence quenching of free indole and 7-azaindole are compared. The fluorescence quenching of octapeptides containing both chromophores is also compared. It is the thesis of this article that the nonexponential fluorescence decay of the 7-azatryptophan-containing octapeptide is a consequence of excited-state tautomerization of the 7-azaindole chromophore. This tautomerization is suggested to be promoted by solvent reorganization induced by the peptide backbone or by direct interactions of the 7-azaindole with neighboring amino acid side chains.     

Energy migration in novel pH-triggered self-assembled beta-sheet ribbons

Energy migration between tryptophan residues has been experimentally demonstrated in self-assembled peptide tapes. Each peptide contains 11 amino acids with a Trp at position 6. The peptide self-assembly is pH-sensitive and forms amphiphilic tapes, which further stack in ribbons (double tapes) and fibrils in water depending on the concentration. Fluorescence spectra, quenching, and anisotropy experiments showed that when the pH is lowered from 9 to 2, the peptide self-assembly buries the tryptophan in a hydrophobic and restricted environment in the interior of stable ribbons as expected on the basis of the peptide design. These fluorescence data support directly and for the first time the presence of such ribbons which are characterized by a highly packed and stable hydrophobic interior. In common with Trp in many proteins, fluorescence lifetimes are nonexponential, but the average lifetime is shorter at low pH, possibly due to quenching with neighboring Phe residues. Unexpectedly, time-resolved fluorescence anisotropy does not change significantly with self-assembly when in water. In highly viscous sucrose-water mixtures, the anisotropy decay at low pH was largely unchanged compared to that in water, whereas at high pH, the anisotropy decay increased significantly. We concluded that depolarization at low pH was not due to rotational diffusion but mainly due to energy migration between adjacent tryptophan residues. This was supported by a master equation kinetic model of Trp-Trp energy migration, which showed that the simulated and experimental results are in good agreement, although on average only three Trp residues were visited before emission.     

THE PHOTOPHYSICAL PROBE, 7-AZATRYPTOPHAN, IN SYNTHETIC PEPTIDES

7-Azatryptophan is proposed as an alternative to tryptophan as a photophysical probe in the study of protein structure and dynamics. Not only are the spectral characteristics of 7-azatryptophan easily distinguishable from those of tryptophan, but this nonnatural amino acid is shown to be amenablc to incorporation into peptides. We present the first synthesis and purification of a synthetic peptide containing 7-azatryptophan, NAc-Pro-7-azatryptophan-Asn-NH₂, which is shown to be a competitive inhibitor of α-chymotrysin.     

Single-walled carbon nanotube binding peptides: probing tryptophan's importance by unnatural amino acid substitution

Peptides selected from phage-displayed libraries have been found to exhibit high-affinity binding to carbon nanotubes including single-walled carbon nanotubes (SWNTs), multi-walled carbon nanotubes, and single-walled carbon nanohorns. One unique feature of these peptides is that their amino acid sequences are rich in tryptophan and histidine residues. The aim of this study was to investigate the importance of the tryptophan residue in a newly identified SWNT-binding peptide, UW-1, which contains the motif, XTHXXPWTX, where X is any amino acid. Tryptophan was altered in the following ways: mutation to alanine or substitution with three unnatural tryptophan analogues, i.e., 5-fluorotryptophan, 5-hydroxytryptophan, and 7-azatryptophan. Analysis of experimental and computational data suggests that the highest occupied molecular orbital of the tryptophan residue in the peptide interacts with the lowest unoccupied molecular orbital from the SWNT. This information should be important in permitting modulation of peptide affinities to these nanomaterials.     

DEUTERIUM-ISOTOPE EFFECT ON THE FLUORESCENCE YIELDS AND LIFETIMES OF INDOLE DERIVATIVES–INCLUDING TRYPTOPHAN AND TRYPTAMINE

Abstract— The fluorescence yields and lifetimes of indole, five of its alkyl detivatives, tryptophan, and tryptamine have been determined in degassed, heavy and light water at room temperature. All of the compounds have radiative lifetimes nearly identical to the parent compound indole, and a comparison of these results with recently reported data on tryptophyl derivatives disclosed a striking uniformity in radiative lifetimes between indole and many amino acids and peptides which contain the indole group as the fluorescence unit. The fluorescence rate k _f in H₂O, was found to be 4.5 × 10⁷ sec^-1. The nonradiative decay rates were found to vary between 5.1 and 46 × 10⁷ sec^-1 and from a study of the deuterium-solvent isotope effect and the deuterium-substituent effect a mechanism for nonradiative deactivation is proposed which includes an isotopically dependent proton transfer and a pathway involving energy loss via the ring carbon hydrogen vibrations. Tryptophan at pH 7 was found to have a unique nonradiative decay scheme not evidenced at a pH 1 or pH 10.     

Low temperature luminescence behaviours of 7-azatryptophan, 5-hydroxytryptophan and their chromophoric moieties

The non-natural amino acids 7-azatryptophan (7AT) and 5-hydroxytryptophan (5HT) have come into significant recent prominence as novel intrinsic luminescence probes for protein structure, function and dynamics. Here, we examine the low temperature luminescence behaviours of these molecules and their respective chromophoric moieties 7-azaindole (7AI) and 5-hydroxyindole (5HI) in representative solvent media. To ascertain, in particular, the potential usefulness of 7AT and 5HT as phosphorescence probes for exploring protein environments with different hydrogen bonding characteristics, a comparison is made of the phosphorescence properties of 7AI and 5HI chromophores in frozen solutions of ethanol and ethyl acetate at 77 K. These solvent media have been chosen as representative models for polar protic and aprotic environments in proteins, respectively. Our findings indicate that one or more of the phosphorescence emission parameters (phosphorescence emission maxima, relative yield and phosphorescence lifetime) of 7AI and 5HI chromophores can serve as sensitive and discriminating probes of hydrogen bonding and related aspects of their surrounding environments. Furthermore, in a model viscous environment (glycerol at low temperatures) significant temperature dependence and red edge excitation shift (REES) effects are observed for the fluorescence emission of 7AT and its chromophoric moiety 7AI. This is consistent with pronounced dipolar relaxation properties of these molecules, and suggests interesting possibilities for exploiting REES in exploring their environmental rigidity in motionally constrained situations.     

PHOSPHORESCENCE MAXIMA AND TRIPLET STATE LIFETIMES OF NAD + AND ε-NAD+ IN TERNARY COMPLEXES WITH HORSE LIVER ALCOHOL DEHYDROGENASE

This paper describes the phosphorescence emission and decay times of NAD+ and its fluorescent etheno derivative, epsilon-NAD+, in the pyrazole ternary complex with horse liver alcohol dehydrogenase (ADH). We show that the epsilon-NAD+ triplet state, as well as the tryptophan triplet state, can be utilized to monitor the coenzyme-enzyme interaction. The decays of NAD+ and AMP are single exponential, and the lifetimes are the same within experimental error. The phosphorescence lifetimes, evaluated as single exponentials, are slightly shorter in epsilon-NAD+ than they are in epsilon-AMP. Whereas the decay of epsilon-AMP was adequately fit by a single exponential with a time constant of very close to 0.5 s, it was necessary to fit the decay of epsilon-NAD+ to a double exponential. Ternary complexes with NAD+ excited at 297 nm exhibit decay kinetics nearly identical to those of ADH by itself. On the other hand, when excitation of the epsilon-NAD+ ternary complex is provided at 313 nm, where there is very little absorption by either tryptophan residue, the decay law of the ternary complex is similar to that of epsilon-NAD+ in solution. Our results demonstrate that NAD+ and epsilon-NAD+ quench tryptophan phosphorescence in ADH. Normalizing the phosphorescence intensity to the 0-0 vibronic band assigned to Trp-15 (blue-edge), we calculate a 21% decrease in the phosphorescence associated with Trp-314 at stoichiometric saturation of the coenzyme binding sites with NAD+ in the ternary complex. When the active sites are saturated with epsilon-NAD+, the relative phosphorescence due to Trp-314 decreases by 63%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultraviolet laser induced fluorescence of human aorta

Laser induced fluorescence from normal human aorta is studied with u.v. excitations of 305 to 310 nm, observing emission from 320 to 500 nm. In this region LIF lineshapes are strongly dependent on the excitation wavelength, suggesting that at least two fluorophores are being observed. The short wavelength fluorophore, peaking at 34Onm, is identified as tryptophan, while the longer wavelength fluorophore, peaking at 387 nm, is associated with collagen and elastin. In addition, fluorescence time decays of each component are measured with a time correlated photon counting system. A four-exponential fit of each decay is necessary to extract fluorescence lifetimes, which range from 33 ps to 8.6 ns.     

Luminescence behaviour of 5-hydroxyindole in different environments

Steady state fluorescence emission spectroscopic studies along with some lifetime measurements have been performed for 5-hydroxyindole (5HI) in different environments. 5HI merits particular attention, since it is the chromophoric moiety of the non-natural amino acid 5-hydroxytryptophan (5HT), which has come into significant, recent prominence as a novel intrinsic optical probe for protein structure, function and dynamics. Studies in representative homogeneous solvents and solvent-mixtures indicate that unlike other fluorophores of related interest like indole (I) and 7-azaindole (7AI), the fluorescence emission maximum (lambda(em)max) of 5HI is relatively insensitive to solvent polarity. This behaviour suggests the lack of appreciable solvent dipolar relaxation in 5HI, which is consistent with our low temperature (77 K) emission data. Notwithstanding such limitation, fluorescence anisotropy (r) and quenching studies are shown to be effective for exploring changes in the micro-environments of 5HI in sodium bis-(2-ethylhexyl)sulfosuccinate (AOT) reverse micellar assemblies (which serve as a biomembrane mimetic model system) with variation in water/surfactant molar ratio (w0).     

Time-resolved fluorescence of the bacteriophage T4 capsid protein gp23

The time-resolved fluorescence properties of the bacteriophage T4 capsid protein gp23 are investigated. The structural characteristics of this protein are largely unknown and can be probed by recording time-resolved and decay-associated fluorescence spectra and intensity decay curves using a 200 ps-gated intensified CCD-camera. Spectral and decay data are recorded simultaneously, which makes data acquisition fast compared to time-correlated single-photon counting. A red-shift of the emission maximum within the first nanosecond of decay is observed, which can be explained by the different decay-associated spectra of fluorescence lifetimes of the protein in combination with dipolar relaxation. In addition, iodide quenching experiments are performed, to study the degree of exposure of the various tryptophan residues. A model for the origin of the observed lifetimes of 0.032 +/- 0.003, 0.39 +/- 0.06, 2.1 +/- 0.1 and 6.8 +/- 0.8 ns is presented: the 32 ps lifetime can be assigned to the emission of a buried tryptophan residue, the 0.4 and 2.1 ns lifetimes to two partly buried residues, and the 6.8 ns lifetime to a single tryptophan outside the bulk of the folded gp23.     

On the heterogeneity of fluorescence lifetime of room temperature ionic liquids: onset of a journey for exploring red emitting dyes

An excitation and emission wavelength dependent non-exponential fluorescence decay behaviour of room temperature ionic liquids (RTILs) has been noted. Average fluorescence lifetimes have been found to vary by a factor of three or more. Red emitting dyes dissolved in RTILs are found to follow hitherto unobserved single exponential fluorescence decay behaviour.     

THE FLUORESCENCE OF TRYPTOPHYL PEPTIDES

Abstract— Tryptophan lifetimes and relative quantum yields have been determined for a group of small (1–4 residues) peptides and peptide hormones [luteinizing releasing factor. mellitin, glucagon. glucagon 22–29, glucagon 1–26. glucagon 1–27 (homoserine)]. All of the larger peptides and most of the smaller peptide anions exhibit nonexponential decay. Peptide quenching in the small peptides is more effective when the bonding is at the amino rather than at the carboxyl end of tryptophan. With the exception of tryptophylglycine. quenching by NH⁺₃ is thought not to involve proton transfer. The results suggest that a decay component of 3–4 ns is expected whenever large peptides and proteins contain a solvent exposed tryptophan.     

(Sub)-picosecond spectral evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system

The spectral evolution of three photoactive proteins has been investigated by measuring the fluorescence with good temporal and wavelength resolution and a high signal-to-noise ratio. Upon excitation at 400 nm wild-type (wt) PYP both at neutral pH and in the low-pH blueshifted pBdark state exhibited a strong quenching of the fluorescence, the major part of which could be described by lifetimes of about 1.7 and 7.7 ps. The remaining fluorescence decay occurred multiexponentially with lifetimes between 30 and 125 ps. Additionally, in wtPYP at neutral pH, a dynamic Stokes shift was found to occur with a time constant of about 0.25 ps. In a PYP preparation that was reconstituted with the chromophore 7-hydroxy-coumarin-3- carboxylic acid rather than the native coumaric acid, and which is therefore not capable of performing the cis-trans-isomerization that initiates the photocycle in wtPYP, the fluorescence was found to decay multiexponentially with lifetimes of 51 ps, 0.33 and 3.77 ns. Additionally, dynamic Stokes shifts were observed with time constants of about 0.1 and 3.5 ps. Upon comparison of the dynamics of this preparation with that of wtPYP the multiexponential decay with lifetimes of 1.7 and 7.7 ps found in wtPYP was attributed to photochemistry of the p-coumaric-acid chromophore. The emission from bacteriorhodopsin mutant D85S upon excitation at 635 nm decays biexponentially with estimated lifetimes of 5.2 and 19.1 ps. No dynamic Stokes shift was observed here. Four lifetimes were needed to describe the decay of the emission from the A* state in the green fluorescent protein. From a target analysis it was concluded that the longer lifetimes are accompanied by a decreasing probability of forming I*, which approaches zero with the longest A* lifetime of 1.5 ns. These observations may be explained by heterogeneity of A and by relaxation of A*. In all three systems studied, multiexponential decay of emission was present, suggesting that heterogeneity is a common feature of these chromophore protein complexes.     

Ionization potentials of fluoroindoles and the origin of nonexponential tryptophan fluorescence decay in proteins

This work reports an explanation for the unusual monoexponential fluorescence decay of 5-fluorotryptophan (5FTrp) in single-Trp mutant proteins [Broos, J.; Maddalena, F.; Hesp, B. H. J. Am. Chem. Soc. 2004, 126, 22-23] and substantially clarifies the origin of the ubiquitous nonexponential fluorescence decay of tryptophan in proteins. Our results strongly suggest that the extent of nonexponential fluorescence decay is governed primarily by the efficiency of electron transfer (ET) quenching by a nearby amide group in the peptide bond. Fluoro substitution increases the ionization potential (IP) of indole, thereby suppressing the ET rate, leading to a longer average lifetime and therefore a more homogeneous decay. We report experimental IPs for a number of substituted indoles including 5-fluoroindole, 5-fluoro-3-methylindole, and 6-fluoroindole, along with accurate ab initio calculations of the IPs for these and 20 related molecules. The results predict the IP of 5-fluorotryptophan to be 0.19 eV higher than that of tryptophan. 5-Fluoro substitution does not measurably alter the excitation-induced change in permanent dipole moment nor does it change the fluorescent state from 1La to 1Lb. In combination with electronic structure information this argues that the increased IP and the decreased excitation energy of the 1La state, together 0.3 eV, are solely responsible for the strong reduction of electron transfer quenching. 6-Fluoro substitution is predicted to increase the IP by a mere 0.09 eV. In agreement with our conclusions, the fluorescence decay curves of 6-fluorotryptophan-containing proteins are well fit using only two decay times compared to three required for Trp.     

Probing folded and unfolded states of outer membrane protein a with steady-state and time-resolved tryptophan fluorescence

Steady-state and time-resolved fluorescence measurements on each of five native tryptophan residues in full-length and truncated variants of E. coli outer-membrane protein A (OmpA) have been made in folded and denatured states. Tryptophan singlet excited-state lifetimes are multiexponential and vary among the residues. In addition, substantial increases in excited-state lifetimes accompany OmpA folding, with longer lifetimes in micelles than in phospholipid bilayers. This finding suggests that the Trp environments of OmpA folded in micelles and phospholipid bilayers are different. Measurements of Trp fluorescence decay kinetics with full-length OmpA folded in brominated lipid vesicles reveal that W102 is the most distant fluorophore from the hydrocarbon core, while W7 is the closest. Steady-state and time-resolved polarized fluorescence measurements indicate reduced Trp mobility when OmpA is folded in a micelle, and even lower mobility when the protein is folded in a bilayer. The fluorescence properties of truncated OmpA, in which the soluble periplasmic domain is removed, only modestly differ from those of the full-length form, suggesting similar folded structures for the two forms under these conditions.     

Lifetimes of partial charge transfer exciplexes of 9-cyanophenanthrene and 9-cyanoanthracene

The fluorescence decays of several exciplexes with partial charge transfer have been investigated in solvents of various polarity. The measured lifetimes are found to be in reasonable agreement with the activation enthalpy and entropy of exciplex decay obtained earlier from the temperature dependence of the exciplex emission quantum yields. For exciplexes with 9-cyanophenanthrene substantial contribution of the higher local excited state into the exciplex electronic structure is found and borrowed intensity effect enhances the exciplex emission rate constants.     

Fluorescence reabsorption in anthracene single crystals: Lifetime variations with emission wavelength and temperature

Fluorescence spectra and lifetimes of anthracene melt-grown single crystals and sublimation flakes have been examined at 298 and 77°K, using a mono-photon counting technique for the lifetime measurements. The observed emission decay times were nearly independent of the excitation wavelength, though a small dependence of the fluorescence spectrum on the excitation wavelength was noted. By contrast, large variations of fluorescence lifetimes in thick crystals were found as a function of emission wavelength. For thick melt-grown single crystals at 298°K the lifetime was found to increase from 9.8 nsec at 405 nm to 20.4 nsec at 445 nm. For sublimation flakes at 77°K and at 298°K and for thick melt-grown crystals at 77°K, the lifetimes were less than 10 nsec and were nearly independent of emission wavelength. Despite these relatively large variations in lifetimes, the decay rates at each separate wavelength remained exponential, within experimental error. Theoretical calculations were made of emission lifetimes based on a model with one reabsorbing state. The calculations are in substantial agreement with the experimental results.