Fluorescence Decay Of Tryptophan Research Articles

Copper(II) Binding to the Intrinsically Disordered C-Terminal Peptide of SARS-CoV-2 Virulence Factor Nsp1.

The first encoded SARS-CoV-2 protein (Nsp1) binds to the human 40S ribosome and blocks synthesis of host proteins, thereby inhibiting critical elements of the innate immune response. The final 33 residues of the natively unstructured Nsp1 C-terminus adopt a helix-turn-helix geometry upon binding to the ribosome. We have characterized the fluctuating conformations of this peptide using circular dichroism spectroscopy along with measurements of tryptophan fluorescence and energy transfer. Tryptophan fluorescence decay kinetics reveal that copper(II) binds to the peptide at micromolar concentrations, and electron paramagnetic resonance spectroscopy indicates that the metal ion coordinates to the lone histidine residue.

Effect of Dinitrosyl Iron Complex on Albumin Conformation.

Binding of dinitrosyl iron complex (DNIC) to albumin was studied using time-resolved fluorescence (TRF) and electron spin resonance (ESR) spectroscopy. It was found that the fluorescence lifetime of bovine serum albumin (BSA) and human serum albumin (HSA) decreases with binding and depends on DNIC concentration. The observed biexponential pattern of the BSA tryptophan (Trp) fluorescence decay is explained by the presence of two tryptophan residues in the protein molecule. We believe that DNIC forms stable complexes with the cysteine (Cys34) residue in the domain I of albumin. It was shown that the lifetime of albumin tryptophan fluorescence decreased during co-incubation of BSA with DNICs and glutathione. Effects of DNIC on the binding of specific spin-labeled fatty acids with albumin in human blood plasma were studied in vitro. The presence of DNIC in blood plasma does not change conformation of albumin domains II and III. We suggest that the most possible interaction between DNICs and albumin is the formation of a complex; and nitrosylation of the cysteine residue in the albumin domain I occurs without the changes in albumin conformation.

Interaction of an IκBα Peptide with 14-3-3.

Inflammatory responses mediated by the transcription factor nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) play key roles in immunity, autoimmune diseases, and cancer. NF-κB is directly regulated through protein–protein interactions, including those with IκB and 14-3-3 proteins. These two important regulatory proteins have been reported to interact with each other, although little is known about this interaction. We analyzed the inhibitor of nuclear factor kappa B α (IκBα)/14-3-3σ interaction via a peptide/protein-based approach. Structural data were acquired via X-ray crystallography, while binding affinities were measured with fluorescence polarization assays and time-resolved tryptophan fluorescence. A high-resolution crystal structure (1.13 Å) of the uncommon 14-3-3 interaction motif of IκBα (IκBαpS63) in a complex with 14-3-3σ was evaluated. This motif harbors a tryptophan that makes this crystal structure the first one with such a residue visible in the electron density at that position. We used this tryptophan to determine the binding affinity of the unlabeled IκBα peptide to 14-3-3 via tryptophan fluorescence decay measurements.

Time-Resolved Tryptophan Fluorescence as an Indicator of Alterations in Serum Proteins in Melancholic Depression

Abstract—The goal of this work was to search for blood serum parameters that would be associated with the state of patients with mental disorders. Such indicators are needed for an objective assessment of this state versus the prevailing subjective evaluation methodologies. The kinetics of tryptophan fluorescence decay in the serum albumin fraction was compared in patients with melancholic depression (before treatment) and in healthy volunteers. Albumin fluorescence is mainly due to the tryptophan 214 residue, which is located in the immediate vicinity of the first drug-binding center of the molecule. The decay kinetics were described as a sum of three exponential functions with lifetimes τi (in the 6.5, 2.8 and 1.0 ns region) and the amplitudes Ai. The τi values were similar in both groups of individuals. In contrast, there was a significant difference between patients and controls in the A1/A3 amplitude ratio. It is suggested that the A1/A3 value can be considered as a potential marker indicating the presence or absence of melancholic depression in patients before treatment.

Temperature Dependence of Tryptophan Fluorescence Lifetime in Aqueous Glycerol and Trehalose Solutions.

The temperature dependences of tryptophan fluorescence decay kinetics in aqueous glycerol and 1M trehalose solutions were examined. The fluorescence decay kinetics were recorded in the spectral region of 292.5-417.5nm with nanosecond time resolution. The kinetics curves were approximated by the sum of three exponential terms, and the spectral distribution (DAS) of these components was determined. An antisymbatic course of fluorescence decay times of two (fast and medium) components in the temperature range from-60 to+10°C was observed. The third (slow) component showed only slight temperature dependence. The antisymbatic behavior of fluorescence lifetimes of the fast and medium components was explained on the assumption that some of the excited tryptophan molecules are transferred from a short-wavelength B-form with short fluorescence lifetime to a long-wavelength R-form with an intermediate fluorescence lifetime. This transfer occurred in the indicated temperature range.

Fluorescence kinetics of tryptophan in a heterogeneous environment

The potentially highly informative, but complex fluorescence decay of amino acids in protein is not fully understood and presents a barrier to understanding. Here we have tested a new and general approach to describing experimentally measured the fluorescence decay in a heterogeneous macroscopic sample. The decay parameters carry information on the features of the kinetics induced by the environment’s heterogeneity. Bayesian interference demonstrated that the model fits well to the fluorescence decay of tryptophan in human serum albumin (HSA). The approach has the potential to accelerate photophysical research of heterogeneous media and, specifically, to solve a critical outstanding problem in interpreting protein fluorescence, paving the way to further progress in biomedical research.

Computational Predictions of Exponential and Non-Exponential Tryptophan Fluorescence Decay in NATA, the Villin Headpiece Subdomain, and other Proteins

Decay of fluorescence from a single Tryptophan (Trp) in a peptide or protein is sometimes exponential and sometimes non-exponential. Understanding the details of this behavior has proven elusive. We have had qualitative success predicting average fluorescence quantum yields for a variety of proteins based on the stabilization of the charge transfer (CT) state arising from electron transfer from the aromatic ring to a backbone amide by the protein electrostatic environment. Here we report 100ns - 1 microsecond MD simulations, augmented by quantum mechanical (ZINDO) computations of the fluorescing and CT states, on N-acetyl-tryptophan-amide (NATA) and 20 single Trp proteins, including the highly studied fast folding villin headpiece. Although the environment for NATA in water is very similar to that of solvent exposed Trps on the surface of a protein, which almost always exhibit non-exponential decay, NATA shows a surprisingly pure single-exponential decay. In our simulations, all possible rotamer states are well represented, and transitions between rotamers happen at a rate of about 1 per 5 ns. Preliminary results indicate that rare conformations in which an amide carbonyl is H-bonded by 2-3 waters produce spikes of high quenching, more or less independent of rotamer state. Survival curve averaging of the 600 ns trajectory yields a single exponential decay of near 3 ns. For folded villin at 300 K, we find that rotamer transitions on Trp occur only every ∼100 ns and that quenching by the nearby His+ happens only during these transient events, although the helix is always intact. Villin is therefore predicted to show extreme heterogeneity in lifetimes. This provides a mechanism for Trp fluorescence to report the global folding rate. Heterogeneity for the other proteins will also be discussed.

Identification of tyrosine in the presence of tryptophan using Cd 2+-enriched colloidal CdS nanoparticles: A fluorescence spectroscopic study

A simpler identification method of tyrosine in the presence of tryptophan using CdS nanoparticles by conventional spectroscopic technique is proposed. Effect of both sulfide-enriched CdS as well as Cd 2+-enriched CdS on tryptophan is investigated through absorption and emission spectroscopy. Quenching of tryptophan emission obeyed Stern–Volmer relation and was found to be independent of temperature, indicating a possible static quenching. The time-resolved fluorescence decay of tryptophan was minimally affected by sulfide-enriched CdS as well as Cd 2+-enriched CdS nanoparticles, suggesting quenching to be static. In the presence of Cd 2+-enriched CdS nanoparticles, the emission of tryptophan in phosphate buffer shows a typical spectral broadening along with a long wavelength increase in fluorescence emission. Additionally, spectra followed a typical isoemissive point at 440 nm when tryptophan alone was there. Similarly, isoemissive point at 340 nm was observed in the case of tyrosine. However, a further red shift of isoemissive point (470 nm) in the mixture of both tyrosine and tryptophan was observed. This observation might make Cd 2+-enriched CdS nanoparticles useful for using as marker for tyrosine in the presence of tyrptophan.

Selective excitation of tryptophan fluorescence decay in proteins using a subnanosecond 295nm light-emitting diode and time-correlated single-photon counting

We demonstrate an AlGaN light-emitting diode (LED) giving pulses of ∼600ps full width half maximum, 0.35μW average power, 0.6mW peak power, and ∼12nm bandwidth at 295nm. This source is ideal for protein intrinsic tryptophan fluorescence decay research without the unwanted excitation of tyrosine and paves the way to lab-on-a-chip protein assays using fluorescence decay times. Fluorescence decay and anisotropy decay measurements of human serum albumin are reported and the usefulness of the 295nm LED demonstrated in comparisons with a nanosecond flashlamp and LEDs with nominal wavelength emission of 280nm.

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.

In vivo synthesized proteins with monoexponential fluorescence decay kinetics.

Tryptophan, when in a protein, typically shows multiexponential fluorescence decay kinetics. Complex kinetics prevents a straightforward interpretation of time-resolved fluorescence protein data, particularly in anisotropy studies or if the effect of a dynamic quencher or a resonance energy transfer (RET) acceptor is investigated. Here, time-resolved fluorescence data are presented of an isosteric tryptophan analogue, 5-fluorotryptophan, which when biosynthetically incorporated in proteins shows monoexponential decay kinetics. Data are presented indicating that the presence of a fluoro atom at the 5-position suppresses the electron transfer rate from the excited indole moiety to the peptide bond. This process has been related to the multiexponential fluorescence decay of tryptophan in proteins. The monoexponential decay of 5-fluorotryptophan makes it possible to measure simultaneously multiple distances between 5-fluorotryptophan and a RET acceptor. We demonstrate that for an oligomeric protein, consisting of two single-tryptophan-containing subunits, the individual distances between 5-fluorotryptophan and the single substrate binding site can be resolved using a substrate harboring a RET acceptor.

Interpretation of Fluorescence Decays using a Power-like Model

A power-like decay function, characterized by the mean excited-state lifetime and relative variance of lifetime fluctuation around the mean value, was applied in analysis of fluorescence decays measured with the aid of time-correlated single photon counting. We have examined the fluorescence decay, in neutral aqueous medium, of tyrosine ( l-tyrosine and N-acetyl- l-tyrosinamide), and of the tyrosine residues in a tryptophan-free protein, the enzyme purine nucleoside phosphorylase from Escherichia coli in a complex with formycin A (an inhibitor), and orthophosphate (a co-substrate). Tryptophan fluorescence decay was examined in neutral aqueous medium for l-tryptophan, N-acetyl- l-tryptophanamide, and for two tryptophan residues in horse liver alcohol dehydrogenase. To detect solvent effect, fluorescence decay of Nz-acetyl- l-tryptophanamide in aqueous medium was compared with that in dioxan. Hitherto, complex fluorescence decays have usually been analyzed with the aid of a multiexponential model, but interpretation of the individual exponential terms (i.e., pre-exponential amplitudes and fluorescence lifetimes), has not been adequately characterized. In such cases the intensity decays were also analyzed in terms of the lifetime distribution as a consequence of an interaction of fluorophore with environment. We show that the power-like decay function, which can be directly obtained from the gamma distribution of fluorescence lifetimes, is simpler and provides good fits to highly complex fluorescence decays as well as to a purely single-exponential decay. Possible interpretation of the power-like model is discussed.

Ultrafast Polarized Fluorescence Measurements on Tryptophan and a Tryptophan-Containing Peptide

In this work polarized picosecond fluorescence measurements were performed on isolated tryptophan and tryptophan in a small 22-mer peptide using a streak camera coupled to a spectrograph as a detection system. In both cases the fluorescence decay was multiexponential with decay times of ∼500 ps and ∼4 ns. Surprisingly, also a short-lived (∼16 ps) isotropic fluorescence component was found for both tryptophan and the peptide which, to our knowledge, has never been observed before. Fluorescence anisotropy data showed rotational correlation times of 155 ps and 1.5 ns for the peptide, reflecting local and overall peptide dynamics, respectively. Recently it was argued [Lakowicz, J. R. Photochem. Photobiol. 2000, 72, 421] that the nonexponential fluorescence decay of tryptophan in proteins is mainly due to spectral relaxation, whereas for isolated tryptophan it is due to different rotamers. Our results do not support this view. In contrast we conclude in both cases that the different fluorescence decay times should be ascribed to different rotameric states.

Structure–function relationships in the neurotoxin Vipoxin from the venom of Vipera ammodytes meridionalis

The neurotoxic complex Vipoxin is the lethal component of the venom of Vipera ammodytes meridionalis, the most toxic snake in Europe. It is a complex between a toxic phospholipase A 2 (PLA 2) and a non-toxic and catalytically inactive protein, stabilizing the enzyme and reducing the activity and toxicity. Structure–function relationships in this complex were studied by spectroscopic methods. A good correlation between the ionization behaviour and accessible surface area (ASA) of the tyrosyl residues was observed. In the toxic PLA 2 subunit phenolic groups participate in H-bonding network that stabilizes the catalytically and pharmacologically active conformation. The tryptophan fluorescence decay of Vipoxin is well fitted by two exponentials with lifetimes of 0.1 (54%) and 2.5 (46%) ns. W20P, W31P and W31I are located in the interface between the two subunits and participate in hydrophobic interactions stabilizing the complex. Dissociation of the complex leads to a transition of the tryptophans from hydrophobic to hydrophilic environment, which influences mainly τ 2. The longer lifetime is more sensitive to the polarity of the environment. Circular dichroism measurements demonstrate that the two components of the neurotoxin preserve their secondary structure after dissociation of the complex. The results of the spectroscopic studies are in accordance with a mechanism of blockade of transmission across the neuromuscular junctions of the breathing muscles by interaction of a dissociated toxic PLA 2 with a membrane. The loss of toxicity is connected with slight changes in the secondary structure of PLA 2. CD studies also show a substantial contribution of disulfide bonds to the stability of the neurotoxic complex and its components.

Influence of solvents and leucine configuration at position 5 on tryptophan fluorescence in cyclic enkephalin analogues.

The fluorescence decay of tryptophan is a sensitive indicator of its local environment within a peptide or protein. In this study we carried out fluorescence measurements of the tryptophan residue of cyclic enkephalin analogues of a general formula X-c[D-Dab(2)-Gly(3)-Trp(4)-Y(5)] where X = Cbz or H and Y = D- or L-Leu, in four solvents [water, methanol, acetonitrile, and dimethyl sulfoxide (DMSO)]. An analysis of the tryptophan fluorescence decays using a discrete-exponential model indicates that tryptophan fluorescence decay can be described by a double exponential function in all solvents studied. Lifetime distribution analysis yields a bimodal distribution in protic solvents (water and methanol), whereas an asymmetric, unimodal distribution in an aprotic solvent (DMSO) and uni- or bimodal distributions in acetonitrile solution, depending on leucine configuration. The data are interpreted in terms of the rotamer model, in which the modality and the relative proportions of the lifetime components are related to the population distribution of tryptophan chi(1) rotamers about the C(alpha)--C(beta) bond. The chirality of the Leu(5) residue and solvent properties affect the local environment of the tryptophan residue and therefore influence the distribution of side-chain rotamers. These results are consistent with the results of theoretical conformational calculations.

Substrate Binding Changes Conformation of the α-, but Not the β-Subunit of Mitochondrial Processing Peptidase

Lifetime analysis of tryptophan fluorescence of the mitochondrial processing peptidase (MPP) from Saccharomyces cerevisiae clearly proved that substrate binding evoked a conformational change of the α-subunit while presence of substrate influenced neither the lifetime components nor the average lifetime of the tryptophan excited state of the β-MPP subunit. Interestingly, lifetime analysis of tryptophan fluorescence decay of the α-MPP subunit revealed about 11% of steady-state fractional intensity due to the long-lived lifetime component, indicating that at least one tryptophan residue is partly buried at the hydrophobic microenvironment. Computer modeling, however, predicted none of three tryptophans, which the α-subunit contains, as deeply buried in the protein matrix. We conclude this as a consequence of a possible dimeric (oligomeric) structure.

Redox-linked conformational changes in bovine heart cytochrome c oxidase: picosecond time-resolved fluorescence studies of cyanide complex.

Picosecond time-resolved fluorescence studies are carried out on cyanide-inhibited and heat-modified cytochrome c oxidase in aqueous lauryl maltoside surfactant solution, as well as in an aqueous vesicle, to understand the conformational changes associated with electron transfer and proton pumping activity of the enzyme. The tryptophan fluorescence decay profiles follow a four exponential model, which also matches the lifetime maxima obtained in a maximum entropy method analysis. The fast lifetime components are highly affected by the reduction and chemical modification of the enzyme. Changes in these lifetime components are related to the conformational changes in the vicinity of the heme centers of the enzyme. The cyanide-inhibited enzyme in the oxidized form shows a fluorescence decay profile similar to that of the native oxidized form, indicating that the conformational changes due to cyanide binding are very small. However, reduction of the cyanide-inhibited enzyme that leaves cyanide bound heme alpha3 oxidized causes a large increase in the fluorescence lifetimes, which indicates very significant conformational changes due to electron transfer to the dinuclear Cu(A) and heme alpha centers. A comparison of the tryptophan fluorescence decay of various other modified forms of the enzyme leads us to propose that the possible site of conformational coupling is located near heme alpha instead of the binuclear heme alpha3-Cu(B) center.

Theoretical studies of the spectroscopic properties of tryptamine, tryptophan and tyrosine

In order to develop model parameters for simulating the spectroscopic properties of tryptophan and tyrosine in solutions and in proteins, we have carried out Hartree–Fock (HF) and configurational interaction (CI) calculations to study the electronic and spectroscopic properties of these fluorophores. Tryptamine, which is structurally related to tryptophan, has also been studied. The 3-21G basis set was used in most of the calculations and only single excitations were included in the CI calculations. Although calculations at this level of approximations have overestimated the transition energies, they have given transition moments within the range of values obtained from semi-empirical calculations and experimental measurements on indole and several of its derivatives. The calculated transition moments can be used in molecular dynamics simulations to study the fluorescence anisotropy decay (FAD) of these fluorophores. We have also estimated the natural atomic orbital charges of the ground and excited states of tryptamine, tryptophan, and tyrosine. These natural atomic orbital charges can be used in molecular dynamics simulations to study the ground- and excited-state dynamics of these fluorophores. The calculated charge redistribution because of optical excitations of tryptophan at different pHs were consistent with the experimental observations that deprotonated tryptophan had red-shifted absorption and emission spectra. Using AM1 adiabatic potential energy curves and a rotamer model employing an electron-transfer mechanism of fluorescence quenching, we have provided an interpretation of the observed pH-dependent fluorescence decay of tryptophan. The conformational and environmental sensitivity of the electronic and spectroscopic properties of these flurophores has also been examined.

Tryptophan Rotamer Distributions in Amphipathic Peptides at a Lipid Surface

The fluorescence decay of tryptophan is a sensitive indicator of its local environment within a peptide or protein. We describe the use of frequency domain fluorescence spectroscopy to determine the conformational and environmental changes associated with the interaction of single tryptophan amphipathic peptides with a phospholipid surface. The five 18-residue peptides studied are based on a class A amphipathic peptide known to associate with lipid bilayers. The peptides contain a single tryptophan located at positions 2, 3, 7, 12, or 14 in the sequence. In aqueous solution, the peptides are unstructured and a triple-exponential function is required to fit the decay data. Association of the peptides with small unilamellar vesicles composed of egg phosphatidylcholine reduces the complexity of the fluorescence decays to a double exponential function, with a reduced dependence of the preexponential amplitude on peptide sequence. The data are interpreted in terms of a rotamer model in which the modality and relative proportions of the lifetime components are related to the population distribution of tryptophan χ 1 rotamers about the C α -C β bond. Peptide secondary structure and the disposition of the tryptophan residue relative to the lipid and aqueous phases in the peptide-lipid complex affect the local environment of tryptophan and influence the distribution of side-chain rotamers. The results show that measurement of the temporal decay of tryptophan emission provides a useful adjunct to other biophysical techniques for investigating peptide-lipid and protein-membrane interactions.

Probing the relation between protein structure and intrinsic tryptophan fluorescence using superrepressor mutants of thetrp repressor

The wavelength dependence of the intrinsic tryptophan fluorescence lifetime of a series of mutants of thetrp repressor protein was characterized in both the native and the denatured states. These mutants belong to a particular class, called superrepressors, as their phenotype, when expressedin vivo, is to repress transcription at lower concentrations of the corepressor, tryptophan. It has been demonstrated previously that these mutations result in distinct and profound modifications of the structural and dynamic properties of the protein [Reedstrom and Royer (1995)J. Mol. Biol. 253, 266; Reedstromet al. (1996)J. Mol. Biol. 264, 32; Smithet al. (1995)Biochemistry 34, 13183]. The present observations reveal that in the native state, these structural and dynamic modifications result in subtle, yet significant alterations in the intrinsic tryptophan fluorescence decay characteristics. Surprisingly, significant differences in the fluorescence decays between the mutants and the wild-type protein were also observed for the guanidine hydrochloride unfolded states. These results are discussed in terms of the various models which have been proposed to explain the decay properties of tryptophan in proteins.