Fluorescence properties of recombinant tropomyosin containing tryptophan, 5-hydroxytryptophan and 7-azatryptophan

Tropomyosin mutants containing either tryptophan (122W), 5-hydroxytryptophan (5OH122W) or 7-azatryptophan (7N122W) have been expressed in Escherichia coli and their fluorescence properties studied. The fluorescent amino acids were located at position 122 of the tropomyosin primary sequence, corresponding to a solvent-exposed position c of the coiled-coil heptapeptide repeat. The emission spectrum of the probe in each mutant is blue-shifted slightly with respect to that of the probe in water. The fluorescence anisotropy decays are single exponential, with a time constant of 2-3 ns while the fluorescence lifetimes of the probes incorporated into the proteins, in water, are nonexponential. Because tryptophan in water has an intrinsic nonexponential fluorescence decay, it is not surprising that the fluorescence decay of 122W is well described by a triple exponential. The fluorescence decays in water of the nonnatural amino acids 5-hydroxytryptophan and 7-azatryptophan (when emission is collected from the entire band) are single exponential. Incorporation into tropomyosin induces triple-exponential fluorescence decay in 5-hydroxytryptophan and double-exponential fluorescence decay in 7-azatryptophan. The range of lifetimes observed for 5-hydroxyindole and 5-hydroxytryptophan at high pH and in the nonaqueous solvents were used as a base with which to interpret the lifetimes observed for the 5OH122W and indicate that the chromophore exists in several solvent environments in both its protonated and unprotonated forms in 5OH122W.     

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.     

Hydrogen-Bond Free Energy of Local Biological Water

Here, we propose an experimental methodology based on femtosecond-resolved fluorescence spectroscopy to measure the hydrogen (H)-bond free energy of water at protein surfaces under isothermal conditions. A demonstration was conducted by installing a non-canonical isostere of tryptophan (7-azatryptophan) at the surface of a coiled-coil protein to exploit the photoinduced proton transfer of its chromophoric moiety, 7-azaindole. The H-bond free energy of this biological water was evaluated by comparing the rates of proton transfer, sensitive to the hydration environment, at the protein surface and in bulk water, and it was found to be higher than that of bulk water by 0.4 kcal mol⁻¹. The free-energy difference is dominated by the entropic cost in the H-bond network among water molecules at the hydrophilic and charged protein surface. Our study opens a door to accessing the energetics and dynamics of local biological water to give insight into its roles in protein structure and function.     

Domain-specific incorporation of noninvasive optical probes into recombinant proteins

An integrated approach is described that allows the domain-specific incorporation of optical probes into large recombinant proteins. The strategy is the combination of two existing techniques, expressed protein ligation (EPL) and in vivo amino acid replacement of tryptophans with tryptophan (Trp) analogues. The Src homology 3 (SH3) domain from the c-Crk-I adaptor protein has been labeled with a Trp analogue, 7-azatryptophan (7AW), using Escherichia coli Trp auxotrophs. Structural, biochemical, and thermodynamic studies show that incorporation of 7AW does not significantly perturb the structure or function of the isolated domain. Ligation of the 7AW-labeled SH3 domain to the c-Crk-I Src homology 2 (SH2) domain, via EPL, generated the multidomain protein, c-Crk-I, with a domain-specific label. Studies of this labeled protein show that the biochemical and thermodynamic properties of the SH3 domain do not change within the context of a larger multidomain protein. The technology described here is likely to be a useful tool in enhancing our understanding of the behavior of modular domains in their natural context, within multidomain proteins.     

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.     

Chemoenzymatic synthesis of the two enantiomers of 7-azatryptophan

7-Azatryptophan is an unnatural α-amino acid with a very potent fluorescent activity. It is used as a vehicle for probing the structure and dynamics of proteins and peptides. Diastereoselective alkylation, diastereoselective protonation and enzymatic resolution have been tested for preparing enantiomerically pure 7-azatryptophan.     

Fluorescence resonance energy transfer between unnatural amino acids in a structurally modified dihydrofolate reductase

The cleavage of a substrate protein by HIV-1 protease has been monitored in real time by the use of a dihydrofolate reductase fusion protein in which a fluorescence donor and a fluorescence acceptor were introduced into sites flanking the HIV-1 protease cleavage site. The amino acids 7-azatryptophan and dabcyl-1,2-diaminopropionic acid were introduced into specific sites of the DHFR fusion protein in an in vitro protein biosynthesizing system using two misacylated suppressor tRNAs, each of which recognized a specific, unique codon introduced into the mRNA. Excitation of the fluorescence acceptor in the initially expressed protein afforded no light production, consistent with quenching by fluorescence resonance energy transfer. Treatment of the elaborated protein with HIV-1 protease cleaved the protein between the fluorescence donor and acceptor, affording a time-dependent increase in fluorescence that was equal in magnitude to that produced by admixture of a stoichiometric amount of free 7-azatryptophan to the solution containing the intact protein.     

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.     

MICROTUBULE ASSEMBLY INHIBITION BY PORPHYRINS and RELATED COMPOUNDS

Abstract— Fourth-derivative spectrophotometry has been applied to the analysis of the ¹L_a O-O band of the indole chromophore. Using several tryptophan models, it is shown that when this band is the most red-shifted, it produces a minimum at about 300 nm in the fourth-derivative spectrum. In proteins this red shift and the 300-nm minimum results from a charge-perturbation effect acting through space on the indole group of tryptophan residues. It is concluded that fourth-derivative spectrophotometry can be used for monitoring the effect of charges on tryptophan residues     

Fluorescence quenching, time-resolved fluorescence and chemical modification studies on the tryptophan residues of snake gourd (Trichosanthes anguina) seed lectin

Fluorescence quenching and time-resolved fluorescence studies have been performed on the galactose-specific lectin purified from snake gourd (Trichosanthes anguina) seeds, in order to investigate the tryptophan accessibility and environment in the native protein and in the presence of bound ligand. Estimation of the tryptophan content by N-bromosuccinimide modification in the presence of 8 M urea yields four residues per dimeric molecule. The emission spectrum of native lectin in the absence as well as in the presence of 50 mM methyl--d-galatopyranoside (MeGal) shows a maximum around 331 nm, which shifts to 361.8 nm upon reduction of the disulfide bonds and denaturation with 8 M urea, indicating that all four tryptophan residues in the native state of this protein are in a hydrophobic environment. The extent of quenching that is observed is highest with acrylamide, intermediate with succinimide, and low with Cs⁺ and I⁻, further supporting the idea that the tryptophan residues are predominantly buried in the hydrophobic core of the protein. The presence of MeGal (50 mM) affects the quenching only marginally. Time-resolved fluorescence measurements yield bi-exponential decay curves with lifetimes of 1.45 and 4.99 ns in the absence of sugar, and 1.36 and 4.8 ns in its presence. These results suggest that the tryptophan residues are not directly involved in the saccharide binding activity of the T. anguina lectin. Of the four quenchers employed in this study, the cationic quencher, Cs⁺, is found to be a very sensitive probe for the tryptophan environment of this lectin and may be useful in investigating the environment of partially buried tryptophan residues and unfolding processes in other proteins as well.     

Regioselective and stereoselective nucleophilic ring opening reactions of a phenyl-substituted aziridine: enantioselective synthesis of beta-substituted tryptophan, cysteine, and serine derivatives

The asymmetric synthesis of beta-phenyl-substituted cysteine, tryptophan, and serine derivatives was successfully developed. In this approach, the key intermediate, enantiomerically pure 3-phenylaziridine-2-carboxylic ester 7, was prepared from alpha,beta-unsaturated ester 1 by employing the Sharpless asymmetric dihydroxylation. The aziridine 7 was treated with 4-methoxybenzylthiol, indole, and acetic acid to give beta-phenyl-substituted cysteine, tryptophan, and serine, respectively, in a clean S(N)2 type ring opening at the C3 position. This general approach can be used to synthesize a variety of beta-substituted novel amino acids.     

UV light-emitting diode-induced fluorescence detection combined with online sample concentration techniques for capillary electrophoresis.

The application of an ultraviolet (UV) light-emitting diode (LED) to on-line sample concentration/fluorescence detection in capillary electrophoresis (CE) is described. The utility of a UV-LED (peak emission wavelength at 380 nm, approximately 2 mW) for fluorescence detection was demonstrated by examining both a naturally fluorescent (riboflavin) compound and a nonfluorescent compound (tryptophan), respectively. The detection limit for riboflavin was determined to be 0.2 ppm by the normal MEKC mode, which was improved to 3-7 ppb when dynamic pH-junction technique was applied. On the other hand, the detection limit of the tryptophan derivative was determined to be 1.5 ppm using the MEKC mode, which was improved to 3 ppb when the sweeping-MEKC mode was applied. In an analysis of an actual sample, the concentrations of riboflavin in beer, and tryptophan in urine and milk samples were determined, respectively.     

HPLC electrochemical fluorometric detection of amino acids including tryptophan using 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole

It has been shown that by electrochemical oxidation 7-nitrobenzo-2-oxa-1,3-diazole-tryptophan (NBD-T) is converted to fluorophores having the same emission and excitation spectra as those for other NBD-amino acids. NBD-dioxindolylalanine was tentatively assumed to be a main electrochemical oxidation product of NBD-tryptophan. A coulochemical cell placed between an analytical column and a fluorometer showed no detrimental effect on the separation of NBD-amino acids by reversed phase HPLC. Highly sensitive fluorescence detection was achieved for amino and imino acids at 10-100 fmol levels. The detection limit for tryptophan was 50 fmol.     

Triazolinedione protein modification: from an overlooked off-target effect to a tryptophan-based bioconjugation strategy

Labelling of tyrosine residues in peptides and proteins has been reported to selectively occur via a ‘tyrosine-click’ reaction with triazolinedione reagents (TAD). However, we here demonstrate that TAD reagents are actually not selective for tyrosine and that tryptophan residues are in fact also labelled with these reagents. This off-target labelling remained under the radar as it is challenging to detect these physiologically stable but thermally labile modifications with the commonly used HCD and CID MS/MS techniques. We show that selectivity of tryptophan over tyrosine can be achieved by lowering the pH of the aqueous buffer to effect selective Trp-labelling. Given the low relative abundance of tryptophan compared to tyrosine in natural proteins, this results in a new site-selective bioconjugation method that does not rely on enzymes nor unnatural amino acids and is demonstrated for peptides and recombinant proteins.

A new strategy for selective tryptophan modification using triazolinedione (TAD) chemistry at pH 4 is shown on peptides and proteins. Additionally, off-target modification of tryptophan residues during the classical TAD-Y click reaction is uncovered.     

Synthesis of C3-substituted 4-azaindoles: an easy access to 4-azamelatonin and protected 4-azatryptophan

C3-Substituted-4-azaindoles were synthesized from pyridylacetonitriles in a two-step sequence allowing the easy introduction of a range of substituents. This strategy permits the rapid synthesis of 4-azamelatonin and a protected 4-azatryptophan.     

Denaturation of bovine serum albumin under the action of cetyltrimethylammonium bromide, according to data from fluorescence analysis

The tryptophan fluorescence of bovine serum albumin (BSA) in solutions with different concentrations of cationic detergent cetyltrimethylammonium bromide (CTAB) at different pH is investigated, providing information on BSA denaturation under the action of CTAB. It is found that BSA denaturation under the action of CTAB at all of the investigated pH values (3.5–8.0) is a single-stage process, as determined by BSA tryptophan fluorescence quenching, by an increased degree of the BSA tryptophan fluorescence polarization, and by the values of the parameters for the rotational diffusion of BSA molecules in CTAB solutions. It is shown that the cationic detergent CTAB is more efficient for BSA denaturation at pH values higher than the BSA isoelectric point (4.9).     

Novel fluorogenic substrates containing bimane system for microdetermination of angiotensin I converting enzyme

As a sensitive fluorometric assay for the activity of angiotensin converting enzyme, bimane-peptides containing tryptophan, i.e., 1,7-dioxo-2,5,6-trimethyl-1H,7H-pyrazolo [1,2-a]pyrazol-3-yl-methylthiomethylcarbonyl-glycyl (or L-phenylalanyl)-L-tryptophyl-L-leucine (or L-proline), were synthesized and shown to be potent fluorogenic substrates for the micro-determination of angiotensin I converting enzyme activity.     

Determination of Tryptophan in Pharmaceutical Formulations Using a Sequential Injection–Zone Fluidic–Chemiluminescence Tubular Reactor

Tryptophan is an important amino acid for humans with a significant role in cell metabolism. Depletion of tryptophan in the human body may contribute to diseases and development of disorders among the human population. It is, therefore, very important to have a reliable, stable, sustainable, and cost-effective analytical method for the determination of tryptophan. Tryptophan was determined using sequential injection–zone fluidics analysis with luminol–hydrogen peroxide and the Firefly with its unique liquid core waveguide flow-cell design as chemiluminescence tubular reactor with a high-sensitivity photomultiplier tube. This was based on an intense chemiluminescence formation of tryptophan in luminol–hydrogen peroxide inside the tubular reactor for measurement. The chemiluminescence intensity was linear with tryptophan in the range of 1.0?×?10^?6 to 1.0?×?10^?3?mol/L, and the limit of detection was 7.5?×?10^?7?mol/L. The precision for the method was 3.6% (relative standard deviation) for six measurements of 1.0?×?10^?4?mol/L tryptophan. The proposed method has been used to determine tryptophan in pharmaceutical formulations. The system is relatively fast for online assays. Eighty seconds are required to complete one cycle providing a throughput of 45 samples/h. The proposed sequential injection analysis–zone fluidics–chemiluminescence system for the assay of tryptophan in certain specific pharmaceutical capsules is simple, reliable, sustainable, and convenient with relatively low-cost consumption of reagents.     

Determination of indican and tryptophan in normal and uraemic patients by high-performance liquid chromatography with a new electrochemical detector

A simple analytical procedure has been developed for the determination of indican and tryptophan in biological fluids by reversed-phase liquid chromatography using a new electrochemical detector consisting of a tubular anode obtained by moulding graphitized carbon black and polyethylene. The hydrodynamic voltammetry of these compounds has been carried out and it has been found that, by operating in isocratic conditions with phosphate buffer (pH 4.0)-methanol (93:7), the reported compounds can be determined directly. The procedure can be applied for the determination of the free compounds on ultrafiltered serum as well as of their total content on serum deproteinized with methanol. Levels of both compounds in normal and uraemic patients have been measured and the relative ratios between free and total content yield a useful marker for patients with renal disease. The limits of quantitation of indican and tryptophan in serum were 5 and 10 ng/ml, respectively. The within-day assay coefficient of variation for total indican and tryptophan ranged from 3.0 to 3.6% and from 3.8 to 4.1%, respectively. The day-to-day assay coefficient of variation for total indican and tryptophan ranged from 3.4 to 3.7% and from 4.6 to 5.0%, respectively.