Tryptophan Fluorescence Lifetimes Research Articles

Discriminating Benign and Malignant Brain Tumors By Spectral Analysis of Cerebrospinal Fluid

The preliminary study is concerned with an innovative technique for the discrimination of malignant brain tumor from benign with reasonable sensitivity and specificity. The entire study is based on the relative fluorescence intensity and life-time of bio-fluorophores like tryptophan and tyrosine which are then validated as a set of ratio parameters. Resultantly, it was seen that malignancy was characterized by three-fold enhancement of tyrosine. The picosecond-time resolved decay time measurement was done, which exhibited two-fold decrease in decay time for malignant sample. The improved accessibility, with minimal invasiveness to the patient, and lower cost are encouraging aspects of this technique.

Crowding by Poly(ethylene glycol) Destabilizes Chemotaxis Protein Y (CheY).

The prevailing understanding of various aspects of biochemical processes, including folding, stability, intermolecular interactions, and the binding of metals, substrates, and inhibitors, is derived from studies carried out under dilute and homogeneous conditions devoid of a crowding-related environment. The effect of crowding-induced modulation on the structure and stability of native and magnesium-dependent Chemotaxis Y (CheY), a bacterial signaling protein, was probed in the presence and absence of poly(ethylene glycol) (PEG). A combined analysis from circular dichroism, intrinsic and extrinsic fluorescence, and tryptophan fluorescence lifetime changes indicates that PEG perturbs the structure but leaves the thermal stability largely unchanged. Intriguingly, while the stability of the protein is enhanced in the presence of magnesium under dilute buffer conditions, PEG-induced crowding leads to reduced thermal stability in the presence of magnesium. Nuclear magnetic resonance (NMR) chemical shift perturbations and resonance broadening for a subset of residues indicate that PEG interacts specifically with a subset of hydrophilic and hydrophobic residues found predominantly in α helices, β strands, and in the vicinity of the metal-binding region. Thus, PEG prompted conformational perturbation, presumably provides a different situation for magnesium interaction, thereby perturbing the magnesium-prompted stability. In summary, our results highlight the dominance of enthalpic contributions between PEG and CheY via both hydrophilic and hydrophobic interactions, which can subtly affect the conformation, modulating the metal-protein interaction and stability, implying that in the context of cellular situation, structure, stability, and magnesium binding thermodynamics of CheY may be different from those measured in dilute solution.

Tryptophan Fluorescence as a Probe for Conformational Dynamics in Zinc Solute Binding Proteins

Bacterial ATP binding cassette (ABC) transporters are important for the uptake of various nutrients, including essential metals such as zinc. These systems require an extracellular solute binding protein (SBP) to bind the substrate with high affinity and specificity and deliver it to the membrane permease for transport. Generally, substrate binding elicits a change from an open to closed conformation that is likely important for permease recognition and transport. However, crystal structures for the metal‐bound and metal‐free forms of several zinc SBPs show relatively subtle or even negligible conformational changes that accompany metal binding. Thus, solution studies are needed for a thorough understanding of the role of conformational dynamics in substrate binding and transport. Here we investigate conformational states and dynamics in a group of zinc SBPs using tryptophan fluorescence intensity and lifetime as a sensitive reporter for the environment of this residue. Useful changes were observed in homologues of the protein AztC from Paracoccus denitrificans and the human pathogen Citrobacter koseri, whereas fluorescence from Citrobacter koseri ZnuA showed no change in fluorescence behavior upon zinc binding. These data combined with isothermal titration calorimetry (ITC) suggest a dynamic equilibrium of at least two conformational states in the apo form and compensatory changes in the holo form that provide for a significant entropic contribution to zinc binding. Correlation with available crystal structures suggests that the formation of a Trp‐Phe pi‐stacking interaction in AztC that is absent in ZnuA may mediate the observed changes in fluorescence. The conformational dynamics identified here highlight diversity in binding mechanisms for zinc SBPs with relevance to the exploitation of these proteins as potential antibiotic drug targets.

Temperature Dependence of Tryptophan Fluorescence Lifetime as an Indicator of Its Microenvironment Dynamics.

The spectral-kinetic characteristics of the fluorescence of the tryptophan molecule in an aqueous solution and in the composition of a protein (albumin) were studied in the temperature range from -170 to25°C. To explain the observed changes in the spectra and the tryptophan fluorescence lifetime with temperature, a model of transitions between the excited and ground states involving a charge-transfer state was used, which takes into account the nonlinear nature of the dynamics of these transitions. In these processes, an important role is played by the interaction of tryptophan molecules with its microenvironment, as well as rearrangements in the system of hydrogen bonds of the water-protein matrix surrounding the tryptophan molecule.

A Study of the Temperature Dependence of Tryptophan Fluorescence Lifetime in the Range of –170 to +20°С in Various Solvents

A Study of the Temperature Dependence of Tryptophan Fluorescence Lifetime in the Range of –170 to +20°С in Various Solvents

Specific features of the temperature dependence of tryptophan fluorescence lifetime in the temperature range of −170–20 °C

The temperature dependence of tryptophan fluorescence lifetime in an aqueous solution, aqueous solutions of glycerol (50 and 75 %, v/v) and dimethyl sulfoxide (DMSO) (50 and 75 %), and 1 M aqueous trehalose solution was studied in the range of –170 to +20 °C. In this temperature range, the fluorescence kinetics in all samples is best approximated by three exponential terms with characteristic times τ1 ∼ 3 ns, τ2 ∼ 4 ns, and τ3 ∼ 15 ns at room temperature. Temperature dependences of fluorescence lifetimes for the fastest and medium components exhibited antisymbatic (antiphase) behavior in the temperature range of –60 to 10 °C. To explain this behavior, a model was suggested that takes into account the tryptophan (Trp) molecule transition from the excited state to the state with separated charges – charge transfer state (CTS), the transitions back to the excited state, and the radiative transitions of the CTS to the ground state. Mathematically, this model is equivalent to the model that we have suggested earlier in [Gorokhov et al. (2018) Biochemistry (Moscow), 82, 1615–1623] to describe the transitions between different rotamer forms of tryptophan. The obtained results can be used for interpreting the experimental temperature curves of tryptophan fluorescence lifetime in proteins.

Comparison of tryptophan fluorescence lifetimes in cyanobacterial photosystem I frozen in the light and in the dark.

The dependence on temperature of tryptophan fluorescence lifetime in trimeric photosystem I (PSI) complexes from cyanobacteria Synechocystis sp. PCC 6803 during the heating of pre-frozen to - 180°C in the dark or in the light-activated preparations has been studied. Fluorescence lifetime in samples frozen in the light was longer than in samples frozen in the dark. For samples in 65% glycerol at λreg = 335nm and at 20°C, the lifetime of components were as follows: τ1 ≈ 1.2ns, τ2 ≈ 4.9ns, and τ3 ≈ 20ns. The contribution of the first component was negligible. To analyze the contribution of components 2 and 3 derived from frozen-thawed samples, two temperature ranges from - 180 to - 90°C and above - 90°C are considered. In doing so, the contributions of these components appear antiphase course to each other. The dependence on temperature of these contributions is explained by the influence of the microconformational protein dynamics on the tryptophan fluorescence lifetime. In the present work, a comparative analysis of temperature-dependent conformational dynamics and electron transfer in cyanobacterial PSI (Schlodder et al., in Biochemistry 37:9466-9476, 1998) and Rhodobacter sphaeroides reaction center complexes (Knox et al., in J Photochem Photobiol B 180:140-148, 2018) was also carried out.

Polyallylamine hydrochloride coating enhances the fluorescence emission of Human Serum Albumin encapsulated gold nanoclusters

Protein encapsulated gold nanoclusters have received much attention due to the possibility of using them as a non-toxic fluorescent probe or marker for biomedical applications, however one major disadvantage currently is their low brightness and quantum yield in comparison to currently used fluorescent markers. A method of increasing the fluorescence emission of Human Serum Albumin (HSA) encapsulated gold nanoclusters (AuNCs) via a Polyallylamide hydrochloride (PAH) coating is described. PAH molecules with a molecular weight of ~17,500 Da were found to enhance the fluorescence emission of HSA-AuNCs by 3-fold when the protein/polymer concentration ratio is 2:1 in solution. Interestingly, the fluorescence lifetime of the AuNCs was found to decrease while the native tryptophan (TRP) fluorescence lifetime also decreased during the fluorescence emission intensity enhancement caused by the PAH binding. Coinciding with the decrease in fluorescence lifetime, the zeta potential of the system was observed to be zero during maximum fluorescence intensity enhancement, causing the formation of large aggregates. These results suggest that PAH binds to the HSA-AuNCs acting as a linker; causing aggregation and rigidification, which results in a decrease in separation between native TRP of HSA and AuNCs; improving Förster Resonance Energy Transfer (FRET) and increasing the fluorescence emission intensity. These findings are critical to the development of brighter protein encapsulated AuNCs.

The effect of light and temperature on the dynamic state of Rhodobacter sphaeroides reaction centers proteins determined from changes in tryptophan fluorescence lifetime and P+QA− recombination kinetics

The temperature dependencies of the rate of dark recombination of separated charges between the photoactive bacteriochlorophyll and the primary quinone acceptor (QA) in photosynthetic reaction centers (RCs) of the purple bacteria Rhodobacter sphaeroides (Rb. sphaeroides) were investigated. Measurements were performed in water−glycerol and trehalose environments after freezing to −180 °C in the dark and under actinic light with subsequent heating. Simultaneously, the RC tryptophanyl fluorescence lifetime in the spectral range between 323 and 348 nm was measured under these conditions. A correlation was found between the temperature dependencies of the functional and dynamic parameters of RCs in different solvent mixtures.For the first time, differences in the average fluorescence lifetime of tryptophanyl residues were measured between RCs frozen in the dark and in the actinic light. The obtained results can be explained by the RC transitions between different conformational states and the dynamic processes in the structure of the hydrogen bonds of RCs. We assumed that RCs exist in two main microconformations – “fast” and “slow”, which are characterized by different rates of P+ and QA− recombination reactions. The “fast” conformation is induced in frozen RCs in the dark, while the “slow” conformation of RC occurs when the RC preparation is frozen under actinic light. An explanation of the temperature dependencies of tryptophan fluorescence lifetimes in RC proteins was made under the assumption that temperature changes affect mainly the electron transfer from the indole ring of the tryptophan molecule to the nearest amide or carboxyl groups.

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.

Tryptophan Fluorescence Yields and Lifetimes as a Probe of Conformational Changes in Human Glucokinase.

Five variants of glucokinase (ATP-D-hexose-6-phosphotransferase, EC 2.7.1.1) including wild type and single Trp mutants with the Trp residue at positions 65, 99, 167 and 257 were prepared. The fluorescence of Trp in all locations studied showed intensity changes when glucose bound, indicating that conformational change occurs globally over the entire protein. While the fluorescence quantum yield changes upon glucose binding, the enzyme's absorption spectra, emission spectra and fluorescence lifetimes change very little. These results are consistent with the existence of a dark complex for excited state Trp. Addition of glycerol, L-glucose, sucrose, or trehalose increases the binding affinity of glucose to the enzyme and increases fluorescence intensity. The effect of these osmolytes is thought to shift the protein conformation to a condensed, high affinity form. Based upon these results, we consider the nature of quenching of the Trp excited state. Amide groups are known to quench indole fluorescence and amides of the polypeptide chain make interact with excited state Trp in the relatively unstructured, glucose-free enzyme. Also, removal of water around the aromatic ring by addition of glucose substrate or osmolyte may reduce the quenching.

Similarity of decay-associated spectra for tryptophan fluorescence of proteins with different structures

Tryptophan fluorescence lifetimes were analyzed for three proteins: human serum albumin, bovine serum albumin, and bacterial luciferase, which contain one, two, and seven tryptophan residues, respectively. For all of the proteins, the fluorescence decays were fitted by three lifetimes: τ1 = 6–7 ns, τ2 = 2.0–2.3 ns, and τ3 ≤ 0.1 ns (the native state), and τ1 = 4.4–4.6 ns, τ2 = 1.7–1.8 ns, and τ3 ≤ 0.1 ns (the denatured state). Corresponding decay-associated spectra had similar peak wavelengths and spectrum half-widths both in the native state (\(\lambda _{\max }^{{\tau _1}} = 324nm\), \(\lambda _{\max }^{{\tau _2}} = 328nm\), and \(\lambda _{\max }^{{\tau _3}} = 315nm\)), and in the denatured state (\(\lambda _{\max }^{{\tau _1}} = 350nm\), \(\lambda _{\max }^{{\tau _2}} = 343nm\), and \(\lambda _{\max }^{{\tau _3}} = 317nm\)). The differences in the steady-state spectra of the studied proteins were accounted for the individual ratio of the lifetime component contributions. The lifetime components were compared with a classification of tryptophan residues in the structure of these proteins within the discrete states model.

PH-Induced conformational isomerization of leghemoglobin from Arachis hypogea.

The pH dependence of proteins is related to the thermodynamic stability and electrostatic interactions in the native state of a protein. Here we report the pH-induced conformational transition of the heme protein leghemoglobin (Lb) isolated from root nodules of the leguminous plant Arachis hypogea. Unlike the other heme proteins myoglobin, hemoglobin, and cytochrome c, the structural characteristics and interactions of Lb is almost unknown, though its functional importance is already established since it binds oxygen to maintain the environment for N2 fixation. We investigated pH-induced unfolding of this protein and identified a number of conformational isomers using multiple fluorescence observables as a function of pH titration. We have characterized the acid- and base-induced conformational transitions among the structural states over the pH range 2-11. Depending on the solution conditions, Lb can exist in one of three phases: pH 2, 3, 4; pH 5, 6, 7; pH 8, 9, 10. The secondary structure as revealed by CD spectroscopy indicated the maximum percentage of α-helix to be present at pH 7, where the structure of Lb is also most rigid according to fluorescence anisotropy experiments. The fluorescence lifetime of tryptophan was observed to be maximum at pH 10 and minimum at pH 6, suggesting unfolding transitions of Lb. Thus, alteration of the microenvironment of the globin moiety during pH transition ultimately leads to the conformational change of this monomeric protein Lb.

Non-Native Structure Appears in Microseconds during the Folding of E. coli RNase H

The folding pathway of Escherichia coli RNase H is one of the best experimentally characterized for any protein. In spite of this, spectroscopic studies have never captured the earliest events. Using continuous-flow microfluidic mixing, we have now observed the first several milliseconds of folding by monitoring the tryptophan fluorescence lifetime (60μs dead time). Two folding intermediates are observed, the second of which is the previously characterized Icore millisecond intermediate. The new earlier intermediate is likely on-pathway and appears to have long-range non-native structure, providing a rare example of such non-native structure formation in a folding pathway. The tryptophan fluorescence lifetimes also suggest a deviation from native packing in the second intermediate, Icore. Similar results from a fragment of RNase H demonstrate that only half of the protein is significantly involved in this early structure formation. These studies give us a view of the formation of tertiary structure on the folding pathway, which complements previous hydrogen-exchange studies that monitored only secondary structure and observed sequential native structure formation. Our results provide detailed folding information on both a timescale and a size-scale accessible to all-atom molecular dynamics simulations of protein folding.

Origin of Tryptophan Fluorescence Lifetimes. Part 2: Fluorescence Lifetimes Origin of Tryptophan in Proteins

Fluorescence intensity decays of L-tryptophan in proteins dissolved in pH 7 buffer, in ethanol and in 6 M guanidine pH 7.8 and in lyophilized proteins were measured. In all protein conditions, three lifetimes were obtained along the emission spectrum (310-410 nm). The two shortest lifetimes are in the same range of those obtained for L-Trp in water or in ethanol. Thus, these two lifetimes originate from specific two sub-structures existing in the excited state and are inherent to the tryptophan structure independently of the surrounding environment (amino acids residues, solvent, etc.) In proteins, the third lifetime originates from the interactions that are occurring between tryptophan residues and neighboring amino acids. Populations of these lifetimes are independent of the excitation wavelength and thus originate from pre-defined sub structures existing in the excited state and put into evidence after tryptophan excitation. Fluorescence decay studies of different tripeptides having a tryptophan residue in second position show that the best analysis is obtained with two fluorescence lifetimes. Consequently, this result seems to exclude the possibility that peptide bond induces the third fluorescence lifetimes. Indole dissolved in water and/or in ethanol emits with two fluorescence lifetimes that are completely different from those observed for L-Trp. Absence of the third lifetime in ethanol demonstrates that indole behaves differently when compared to tryptophan. Thus, it seems not adequate to attribute fluorescence lifetime or fluorescence properties of tryptophan to indole ring and to compare tryptophan fluorescence properties in proteins to molecules having close structures such as NATA which fluoresces with one lifetime.

Investigation of tryptophan–NADH interactions in live human cells using three-photon fluorescence lifetime imaging and Förster resonance energy transfer microscopy

A method to investigate the metabolic activity of intracellular tryptophan (TRP) and coenzyme-NADH using three-photon (3P) fluorescence lifetime imaging (FLIM) and Förster resonance energy transfer (FRET) is presented. Through systematic analysis of FLIM data from tumorigenic and nontumorigenic cells, a statistically significant decrease in the fluorescence lifetime of TRP was observed in response to the increase in protein-bound NADH as cells were treated with glucose. The results demonstrate the potential use of 3P-FLIM-FRET as a tool for label-free screening of the change in metabolic flux occurring in human diseases or other clinical conditions.

Label Free Fluorescence Lifetime FRET Imaging

Multiphoton microscopy with endogenous contrasts in biological tissues have primarily focused on detecting signals from the reduced nicotinamide adenine dinucleatide (NADH), its dinucleatide phosphate (NADPH), riboflavins, and tryptophan. All these fluorophores have emission in the wavelength range of 400-600 nm. According to an earlier studies on the autofluorescence spectroscopy of ex vivo leukocyte samples under linear (one photon) excitation conditions, signals from the tryptophan (TRP) moieties in cellular proteins should be much stronger than signals from NAD(P)H on the per cell basis. In recent years, considerable interest has been developed in the effect of various diseases upon the metabolic pathway of L-tryptophan and its derivatives. We describe a new method for imaging tumorigenic and non-tumorigenic cell lines by 3-photon exciting of the endogenous protein fluorescence in the ultraviolet (UV) spectral region, where tryptophan is the major fluorophore. Through systematic analysis of FLIM data from live cells, a statistically significant decrease in the fluorescence lifetime of TRP was observed in response to the increase in protein-bound NADH as cells were treated with glucose. Addition of glycolytic substrates significantly quenches tryptophan lifetime. The results demonstrate the potential use of 3P-FLIM-FRET as a tool for label free screening of the change in metabolic flux occurring in human diseases or other clinical conditions.

Biophysical Characterization of Genistein in Its Natural Carrier Human Hemoglobin Using Spectroscopic and Computational Approaches

Steady state and time resolved fluorescence spectroscopy, combined with molecular dynamics simulation, have been used to explore the interactions of a therapeutically important bioflavonoid, genistein, with normal human hemoglobin (HbA). Binding constants estimated from the fluorescence studies were K = (3.5  0.32)  10 4 M −1 for genistein. Specific interactions with HbA were confirmed from flavonoid-induced fluorescence quenching of the tryptophan in the protein HbA. The mechanism of this quenching involves both static and dynamic components as indicated by: (a) increase in the values of Stern-Volmer quenching constants with temperatures, (b) 0  / is slightly > 1 (where 0  and  are the unquenched and quenched tryptophan fluorescence lifetimes (averaged) respectively). Molecular docking and dynamic simulations reveal that genistein binds between the subunits of HbA, ~18 A away from the closest heme group of chain α1, emphasizing the fact that the drug does not interfere with oxygen binding site of HbA.

Complementary Fluorescence and Phosphorescence Study of the Interaction of Brompheniramine with Human Serum Albumin

Binding of the antihistamine drug brompheniramine (BPA) to human serum albumin (HSA) is studied by measuring quenching of the fluorescence and room temperature phosphorescence (RTP) of tryptophan. The modified Stern-Volmer equation was used to derive association constants and accessible fractions from the steady-state fluorescence data. Decay associated spectra (DAS) revealed three tryptophan fluorescence lifetimes, indicating the presence of three HSA conformations. BPA causes mainly static quenching of the long-living, solvent-exposed conformer. RTP spectra and lifetimes, recorded under deoxygenated conditions in the presence of 0.2 M KI, provided additional kinetic information about the HSA-BPA interactions. Fluorescence DAS that were also recorded in the presence of 0.2 M KI revealed that the solvent-exposed conformer is the major contributor to the RTP signal. The phosphorescence quenching is mostly dynamic at pH 7 and mostly static at pH 9, presumably related to the protonation state of the alkylamino chain of BPA. This provides direct insight into the binding mode of the antihistamine drug, as well as kinetic information at both the nanosecond and the millisecond time scales.

Dating bloodstains with fluorescence lifetime measurements.

Dating bloodstains with fluorescence lifetime measurements.