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.     

Tryptophan-Tryptophan Energy Transfer and Classification of Tryptophan Residues in Proteins Using a Therapeutic Monoclonal Antibody as a Model

Intrinsic tryptophan (Trp) fluorescence is often used to determine conformational changes of proteins. The fluorescence of multi-Trp proteins is generally assumed to be additive. This assumption usually holds well if Trp residues are situated at long distances from each other in the absence of any excited state reactions involving these residues and therefore when energy transfer does not occur. Here, we experimentally demonstrate energy transfer among Trp residues and support it by a Master Equation kinetic model applied to a therapeutic monoclonal antibody (mAb). The mAbs are one of the most studied and important biologics for the pharmaceutical industry, and they contain many Trp residues in close proximity. Understanding mAb fluorescence is critical for interpreting fluorescence data and protein-structure relationships. We propose that Trp residues could be categorized into three types of emitters in the mAbs. Experimentally, we categorize them according to solvent accessibility based on dependence of their fluorescence lifetime on the external quencher concentration and their emission wavelength. Theoretically, we categorize with molecular dynamics simulations according to their solvent accessibility. This method of combinatorial mapping of fluorescence characteristics can be utilized to illuminate structural aspects as well as make comparisons of drug formulations for these pharmaceutical proteins.     

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.     

Origin of Fluorescence Lifetimes in Human Serum Albumin. Studies on Native and Denatured Protein

Human serum albumin consists of a single polypeptide of 585 amino acid residues with 1 Trp residue. In the present work, we measured fluorescence lifetimes of the protein in both native and denatured states. The results indicate that Trp emission occurs with three lifetimes in both states. Lifetimes values and contribution to the global emission decay differ between the two states. Data are interpreted as the results of an emission occurring from three substructures of the tryptophan formed in the excited state. Two of these substructures are already present for the tryptophan free in solution. The third lifetime is the result of the interaction between the tryptophan residue and surrounding microenvironment. The populations of these substructures characterized by the pre-exponential parameters of the fluorescence lifetimes are dependent on the fluorophore microenvironment and on the global protein structure.     

New Insights in the Interpretation of Tryptophan Fluorescence

Origin of tryptophan fluorescence is still up to these days a quiz which is not completely solved. Fluorescence emission properties of tryptophan within proteins are in general considered as the result of fluorophore interaction within its environment. For example, a low fluorescence quantum yield is supposed to be the consequence of an important fluorophore–environment interaction. However, are we sure that the fluorophore has been excited upon light absorption? What if fluorophore excitation did not occur as the result of internal conformation specific to the fluorophore environment? Are we sure that all absorbed energy is used for the excitation process? Fluorescence lifetimes of Trp residues are considered to originate from rotamers or conformers resulting from the rotation of the indole ring within the peptide bonds. However, how can we explain the fact that in most of the proteins, the two lifetimes 0.5 and 3 ns, attributed to the conformers, are also observed for free tryptophan in solution? The present work, performed on free tryptophan and tyrosine in solution and on different proteins, shows that absorption and excitation spectra overlap but their intensities at the different excitation wavelengths are not necessarily equal. Also, we found that fluorescence emission intensities recorded at different excitation wavelengths depend on the intensities at these excitation wavelengths and not on the optical densities. Thus, excitation is not equal to absorption. In our interpretation of the data, we consider that absorbed photons are not necessary used only for the excitation, part of them are used to reorganize fluorophore molecules in a new state (excited structure) and another part is used for the excitation process. A new parameter that characterizes the ratio of the number of emitted photons over the real number of photons used to excite the fluorophore can be defined. We call this parameter, the emission to excitation ratio. Since our results were observed for fluorophores free in solution and present within proteins, structural reorganization does not depend on the protein backbone. Thus, fluorescence lifetimes (0.5 and 3 ns) observed for tryptophan molecules result from the new structures obtained in the excited state. Our theory allows opening a new way in the understanding of the origin of protein fluorescence and fluorescence of aromatic amino acids.     

Dipolar Relaxation Times of Tryptophan and Tyrosine in Glycerol and in Proteins: A Direct Evaluation from Their Fluorescence Decays

The dipolar relaxation process induced around tryptophan, indole and tyrosine in viscous media, as well as in several single tryptophan-containing proteins (staphylococcal nuclease, ribonuclease T1, melittin and albumin), has been studied by dynamic fluorescence measurements. A new theoretical model has been developed, including the relaxation dynamics directly in the fluorescence decay function. The phase shift and demodulation data have been fitted with this new algorithm which allows to resolve the different relaxation times influencing the fluorophore excited state. These parameters are in a good agreement with those measured with the traditional time-resolved emission spectroscopy. The results indicate that indeed a correlation exists between the radiative rate change obtained with the new model and the temporal spectral shift reported in the literature. Finally, this new approach has also been extended to the case of superoxide dismutase and phosphofructokinase, allowing to measure the relaxation time even in proteins lacking a temporal spectral shift during the fluorphore's lifetime.     

Steady-State Fluorescence Properties of S. solfataricus Alcohol Dehydrogenase and Its Selenomethionyl Derivative

Alcohol dehydrogenase from the thermoacidophilic Sulfolobus solfataricus (SsADH) is a thermophilic NAD⁺-dependent homotetrameric zinc enzyme whose crystal structure has been recently determined at 1.85 Å resolution by using a selenomethionine-substituted enzyme. In this report the steady-state fluorescence properties of SsADH are related to the two fluorophores Trp95 and 117 located inside the monomer structure and whose indole ring centers are at 5.7 Å to each other. The relatively blue emission of the enzyme (_max = 320 nm) is due to the highly hydrophobic character of the microenvironment determined by six and seven non-polar residues in close contact (<7 Å) with Trp95 and 117, respectively. However, the contribution of the two residues to intrinsic fluorescence appears different since the Trp95 and 117 indole rings are found in the vicinity of five and one polar residue side chains, respectively. The fluorescence intensity of the selenomethionine-substituted enzyme is found to be 40% lower than that of the natural enzyme. Moreover, four out of the nine methionine residues per monomer are found in the vicinity (<6 Å) of as many tyrosine residue side chains, while no methionine-tryptophan interaction is present in the structure. Presumably, selenium acts as a quencher of the nearby tyrosine emission more efficiently than does sulfur, due to its larger electron cloud and polarizability. It cannot be excluded that an effect of selenium is to stabilize the tyrosinate ion allowing a more extended delocalization of the negative charge. Therefore, the decreased tryptophan emission of the seleno-protein would reflect the lower quantum yield of the tyrosine in its ionized state.     

Relation between proteins tertiary structure, tryptophan fluorescence lifetimes and tryptophan S(o)→(1)L(b) and S(o)→(1)L(a) transitions. Studies on α1-acid glycoprotein and β-lactoglobulin

We measured fluorescence lifetimes and fluorescence spectra (excitation and emission) of tryptophan residues of α₁-acid glycoprotein (three Trp residues) and β-lactoglobulin (two Trp residues) in absence and presence of 450 μM progesterone. Progesterone binds only to α₁-acid glycoprotein. In absence of progesterone, each of the two proteins displays three fluorescence lifetimes. Addition of progesterone induces a partial inhibition of the S _o → ¹ L _a transition without affecting fluorescence lifetimes. The same experiments performed in presence of denatured proteins in 6 M guanidine show that addition of progesterone inhibits partially the S _o → ¹ L _a transition and its peak is 15 nm shifted to the red compared to that obtained for native proteins. However, the S _o → ¹ L _b transition position peak is not affected by protein denaturation. Thus, the tertiary structure of the protein plays an important role by modulating the tryptophan electronic transitions. Fluorescence emission decay recorded in absence and presence of progesterone yields three fluorescence lifetimes whether proteins are denatured or not. Thus, protein tertiary structure is not responsible for the presence of three fluorescence lifetimes. These characterize tryptophan substructures reached at the excited states and which population (pre-exponential values) depend on the tryptophan residues interaction with their microenvironment(s) and thus on the global conformation of the protein.     

Phenylalanine fluorescence and phosphorescence used as a probe of conformation for cod parvalbumin

The fluorescence emission and triplet absorption properties of phenylalanine in cod fish parvalbumin type II, a protein that contains no Trp or Tyr, was examined in the time scale ranging from nanoseconds to microseconds at 25°C in aqueous buffer (pH 7.0). In the presence of Ca(II), the decay of fluorescence gave two lifetimes (5.9 and 53 ns) and the triplet lifetime was 425 s. Upon removal of Ca, the fluorescence intensity decreased to values approaching that for free Phe, while the longest fluorescence decay component was 17 ns. At the same time, the decay of triplet showed complex nonexponential kinetics with decay rates faster than in the presence of Ca. Quenching and denaturation analyses suggest that the Phe's are present in a hydrophobic environment in the Ca-bound protein but that the Ca-free protein is relatively unstructured. It is concluded that Phe luminescence in proteins is sensitive to conformation and that the long lifetime of Phe excited states needs to be considered when studying its photochemistry in proteins.     

Sub-Structures Formed in the Excited State are Responsible for Tryptophan Residues Fluorescence in β-Lactoglobulin

Origin of tryptophan residues fluorescence in β-lactoglobulin is analyzed. Fluorescence lifetimes and spectra of β-lactoglobulin solution are measured at pH going from 2 to 12 and in 6 M guanidine. Tryptophan residues emit with three lifetimes at all conditions. Two lifetimes (0.4–0.5 ns and 2–4 ns) are in the same range of those measured for tryptophan free in solution. Lifetimes in the denatured states are lower than those measured in the native state. Pre-exponential values are modified with the protein structure. Data are identical to those already obtained for other proteins. Fluorescence lifetimes characterize internal states of the tryptophan residues (Tryptophan sub-structures) independently of the tryptophan environments, the third lifetime results from the interaction that is occurring between the Trp residues and its environment. Pre-exponential values characterize substructures populations. In conclusion, tryptophan mission occurs from substates generated in the excited state. This is in good agreement with the theory we described in recent works.     

Identification of tyrosine in the presence of tryptophan using Cd-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 Cd²⁺-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 Cd²⁺-enriched CdS nanoparticles, suggesting quenching to be static. In the presence of Cd²⁺-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 Cd²⁺-enriched CdS nanoparticles useful for using as marker for tyrosine in the presence of tyrptophan.     

Fluorescence probes of viscosity: A comparative study of the fluorescence anisotropy decay of perylene and 3,9-dibromoperylene in glycerol

The authors compare the results of fluorescence anisotropy decay measurements for glycerol solutions of perylene with those of 3,9-dibromoperylene (DBP). For both molecules a good linear dependence is observed between the glycerol viscosity (varied by temperature) and the longer rotational correlation time obtained as a result of a global (using data obtained at 256- and 430-nm excitation wavelengths) biexponential analysis of the fluorescence anisotropy decay, at least in the range of 7–60 P for perylene and 4–60 P for DBP. This significantly extends the reported range of 0.5 to 150 cP investigated by Williams and Ben-Amotz [1] with the probe BTBP.     

Tryptophan Residue of the D-Galactose/D-Glucose-Binding Protein from <Emphasis Type="Italic">E. Coli</Emphasis> Localized in its Active Center Does not Contribute to the Change in Intrinsic Fluorescence Upon Glucose Binding

Changes of the characteristics of intrinsic tryptophan fluorescence of the wild type of D-galactose/D-glucose-binding protein from Escherichia coli (GGBPwt) induced by D-glucose binding were examined by the intrinsic UV-fluorescence of proteins, circular dyhroism in the near-UV region, and acrylamide-induced fluorescence quenching. The analysis of the different characteristics of GGBPwt and its mutant form GGBP-W183A together with the analysis of the microenvironment of tryptophan residues of GGBPwt revealed that Trp 183, which is directly involved in sugar binding, has the least influence on the provoked by D-glucose blue shift and increase in the intensity of protein intrinsic fluorescence in comparison with other tryptophan residues of GGBP.     

Dynamics of Trp Residues in Crystals of Human α1-Acid Glycoprotein (Orosomucoid) Followed by Red-Edge Excitation Spectra

The present work reports for the first time the development of a method that allowed us to obtain crystals of orosomucoid complexed to progesterone. Then we investigated the dynamics of the microenvironments of the two buried Trp residues in the crystals of protein, by the red-edge excitation spectra method. The fluorescence excitation spectrum of the crystals is characteristic of that known for Trp residues (_max = 290 nm and bandwidth = 38 ± 1 nm), indicating that the Trp residues are responsible for the fluorescence of the protein in the crystals. The position of the maximum and the bandwidth of the steady-state emission spectrum of the crystals (331 ± 1 and 43 ± 1 nm, respectively) are equal to those obtained in aqueous buffer for the orosomucoid–progesterone complex (330 ± 1 and 43 ± 1 nm) (_ex, 295 nm). Thus, the fluorescence of the crystals occurs from the Trp residues buried in the protein core. The red-edge excitation spectra studies indicate that the Trp residues are surrounded by microenvironments that display motions, a result identical to that observed in solution. Thus, the crystallization process does not modify the structure or the dynamics of orosomucoid core. The fluorescence intensities depend on the angular orientation of the crystals with respect to the polarization of the incident beam. The general feature of this dependence is identical at the three excitation wavelengths used (295, 300, and 305 nm). Our results confirm the fact that the local structure and dynamics are the key for any interpretation of tryptophan fluorescence parameters of orosomucoid.     

Selective fluorescence resonance energy transfer from serum albumins to a bio-active 3-pyrazolyl-2-pyrazoline derivative: A spectroscopic analysis

A novel fluorescent probe and pharmaceutically significant: 3-pyrazolyl-2-pyrazoline derivative (PYZ) has been selected as an acceptor molecule for fluorescence resonance energy transfer (FRET) interaction with serum albumins. Steady state and time resolved fluorescence techniques were applied to elucidate the nature of interaction of PYZ with serum albumins (BSA and HSA). Negligible FRET mediated emission occurred in the case of HSA but an efficient FRET mediated emission resulted in case of BSA. To gain further insight into the FRET selectivity of PYZ with the proteins, FRET from L-tryptophan (donor; native tryptophan) to PYZ (acceptor) was performed with the aim of getting an idea about the steric restrictions imposed on PYZ by the other groups present in BSA and HSA. The studies revealed that the surface bound Trp-134 in BSA allows an efficient FRET process with PYZ while the buried Trp-214 in HSA does not. The unusual selectivity for FRET in case of PYZ and the serum albumins has also been attributed to the complex structure of PYZ due to the presence of bulkier phenyl moieties in it. The complex nature of the excited state photophysics of tryptophan (Trp) in proteins also accounts for this FRET selectivity of PYZ with BSA and HSA.     

Localization and Environment of Tryptophans in Different Structural States of Concanavalin A

We have investigated the localization and environment of tryptophan residues in different quaternary and conformational states (tetrameric, dimeric, monomeric and unfolded) of metallized and demetallized concanavalin A (ConA) by selective chemical modification, fluorescence, and phosphorescence. ConA has four tryptophan residues (Trp 40, Trp 88, Trp 109 and Trp 182) per subunit. The pattern of oxidation by N-bromosuccinimide (NBS) shows that NBS modifies, in dimer, only Trp 182 which remains inaccessible in tetramer, two (Trp 88 along with Trp 182) in monomer, all four in unfolded form in presence of EDTA, and three (possibly Trp 40 along with Trp 88 and Trp 182) in unfolded form from native or remetallized ConA. Utilizing wavelength-selective fluorescence approach, we have observed a red edge excitation shift (REES) of 6–8 nm for tetramer and dimer. A more pronounced REES (11 nm) is observed for oxidized monomer compared to REES (3 nm) for unoxidized species. Acrylamide quenching shows the Stern-Volmer constant (K_SV) for dimer, monomer, unfolded ConA and unfolded apo-ConA being 3.8, 5.2, 12.8, 14.0 M⁻¹, respectively. Phosphorescence studies at 77 K give more structured spectra, with two (0,0) bands at 406.2 (weak) and 413.2 nm for tetramer. However, a single (0,0) band appears at 413.2 for dimer and 412.6 nm for monomer, while the (0,0) band of the oxidized monomer is red shifted to 414.4 nm. These results may provide important insight into subtlety of organization and environment of tryptophans in the context of folding and structural studies of oligomeric proteins including lectins.     

Fluorescence spectra of Rhodamine 6G for high fluence excitation laser radiation

Fluorescence spectral changes of Rhodamine 6G in ethanol and glycerol solutions and deposited as a film on a silica surface have been studied using a wide range of pumping field fluence at 532 nm at room temperature. Blue shift of the fluorescence spectra and fluorescence quenching of the dye molecule in solution are observed at high excitation fluence values. Such effects are not reported for the film sample. The effects are interpreted as the result of population redistribution in the solute-solvent molecular system induced by the high fluence field and the fluence dependence of the radiationless decay mechanism.     

Measuring fast hydrogen exchange rates by NMR spectroscopy

We introduce a method to measure hydrogen exchange rates based on the observation of the coherence of a neighboring spin S such as (15)N that has a scalar coupling J(IS) to the exchanging proton I. The decay of S(x) coherence under a Carr-Purcell-Meiboom-Gill (CPMG) multiple echo train is recorded in the presence and absence of proton decoupling. This method allows one to extract proton exchange rates up to 10(5)s(-1). We could extend the pH range for the study of the indole proton in tryptophan, allowing the determination of the exchange constants of the cationic, zwitterionic, and anionic forms of tryptophan.     

Temporal dependences of dipole moments of prodan molecules

The fluorescence spectra of 6-propyl-2-dimethylaminonaphtalene (prodan), 1-(phenylamino)naphthalene (1-PAN), and 3-aminophthalimide are measured in glycerol with a picosecond resolution. The instantaneous fluorescence spectra and the correlation functions of the time-dependent Stokes shift of the fluorescence spectra of the two latter probes are used to find the time dependence of the dielectric response function of glycerol, which is necessary for calculating the charge transfer kinetics in prodan molecules. Based on the solvatochromism theory and using the experimental dependences of the time-dependent shift of the fluorescence bands for prodan molecules, which are characterized by efficient charge transfer in the excited electronic state, the kinetics of the electric dipole moment of the first singlet state is calculated.