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.     

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.     

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.     

Origin of Tryptophan Fluorescence Lifetimes Part 1. Fluorescence Lifetimes Origin of Tryptophan Free in Solution

Fluorescence intensity decays of L-tryptophan free in polar, hydrophobic and mixture of polar-hydrophobic solvents were recorded along the emission spectrum (310–410 nm). Analysis of the data show that emission of tryptophan occurs with two lifetimes in 100 % polar and hydrophobic environments. The values of the two lifetimes are not the same in both environments while their populations (pre-exponentials values) are identical. Fluorescence lifetimes and pre-exponentials values do not change with the excitation wavelength and thus are independent of excitation energy. Our results indicate that tryptophan emission occurs from two specific sub-structures existing in the excited state. These sub-structures differ from those present in the ground states and characterize an internal property and/or organization of the tryptophan structure in the excited state. By sub-substructure, we mean here tryptophan backbone and its electronic cloud. In ethanol, three fluorescence lifetimes were measured; two lifetimes are very close to those observed in water (0.4–0.5 ns and 2–4 ns). Presence of a third lifetime for tryptophan in ethanol results from the interaction of both hydrophobic and hydrophilic dipoles or chemical functions of ethanol with the fluorophore.     

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.     

Conformational Flexibility of Cytokine-Like C-Module of Tyrosyl-tRNA Synthetase Monitored by Trp144 Intrinsic Fluorescence

The non-catalytic COOH-terminal module formed after proteolytic cleavage of full-length mammalian tyrosyl-tRNA synthetase displays dual function: tRNA binding ability and cytokine activity. With the aim to explore the intramolecular dynamics of C-module in solution we used fluorescence spectroscopy to study conformational changes of isolated protein. We used information from fluorescence spectra and computational model for characterization of a microenvironment of a single tryptophan residue (Trp144). Its fluorescence parameters and protection from quenching by Cs⁺ ions indicate the internal localization—buried into protein globule. The fluorescence quenching of Trp144 by acrylamide suggests rapid conformation dynamics of the C-module in nanosecond time scale. The temperature-induced conformational changes in the C-module were monitored by the fluorescence measurements of Trp144 emission and by red-edge excitation shift. An emission maximum shift up to ∼349 nm and significant decrease of the red-edge shift effect at 37–52 °C indicated a major conformational transition of Trp144 from buried native state into highly relaxing polar solvent environment.     

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.     

Exploration of twisted intramolecular charge transfer fluorescence properties of trans-2-[4-(dimethylamino)styryl]benzothiazole to characterize the protein–surfactant aggregates

The characterization of aggregates of an anionic surfactant, sodium dodecyl sulphate (SDS) with bovine serum albumin (BSA) in various regions of binding isotherm of SDS to BSA with increasing concentration of the former have been done by exploring the twisted intramolecular charge transfer (TICT) fluorescence properties of a probe, trans-2-[4-(dimethylamino)styryl] benzothiazole (DMASBT). The TICT fluorescence, steady-state fluorescence anisotropy and time-resolved fluorescence of DMASBT, and the fluorescence resonance energy transfer (FRET) study reveal the characteristics of the native protein as well as the protein–surfactant aggregates viz., micropolarity, microviscosity, locations of probe, denaturation of protein in various regions of binding isotherm, and also the validation of necklace-bead model. The changes in the polarity and the viscosity of the microenvironment around the probe from one binding region of SDS to other have been reflected in the highly sensitive fluorescence properties of DMASBT. The study of FRET between the DMASBT and the tryptophan residue (Trp) of BSA has identified the locations of the probe molecule in the native protein as well as that in various BSA–SDS aggregates. The energy transfer efficiency decreases, whereas the distance between the DMASBT and the Trp residue increases with increasing concentration of SDS. The significant change in the conformations of protein molecules during the non-cooperative binding region of SDS is evidenced by the fluorescence anisotropic behavior of DMASBT in the same region.     

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.     

Tryptophan Environment and Functional Characterization of a Kinetically Stable Serine Protease Containing a Polyproline II Fold

The single tryptophan residue from Nocardiopsis sp. serine protease (NprotI) was studied for its microenvironment using steady state and time-resolved fluorescence. The emission maximum was observed at 353 nm with excitation at 295 nm indicating tryptophan to be solvent exposed. Upon denaturation with 6 M guanidinum thiocyanate (GuSCN) the emission maxima was shifted to 360 nm. Solute quenching studies were performed with neutral (acrylamide) and ionic (I^- and Cs⁺) quenchers to probe the exposure and accessibility of tryptophan residue of the protein. Maximum quenching was observed with acrylamide. In the native state, quenching was not observed with Cs⁺ indicating presence of only positively charged environment surrounding tryptophan. However; in denatured protein, quenching was observed with Cs⁺, indicating charge reorientation after denaturation. No quenching was observed with Cs⁺ even at pH 1.0 or 10.0; while at acidic pH, a higher rate of quenching was observed with KI. This indicated presence of more positive charge surrounding tryptophan at acidic pH. In time resolved fluorescence measurements, the fluorescence decay curves could be best fitted to monoexponential pattern with lifetimes of 5.13 ns for NprotI indicating one conformer of the trp. Chemical modification studies with phenyl glyoxal suggested presence of Arg near the active site of the enzyme. No inhibition was seen with soyabean trypsin and limabean inhibitors, while, CanPI uncompetitively inhibited NprotI. Various salts from Hofmeister series were shown to decrease the activity and PPII content of NprotI.     

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.     

Fluorescence Characterization of the Hydrophobic Pocket of Cyclophilin B

Human cyclophilin B is a monomeric protein that contains two tryptophan residues, Trp104 and 128. Trp128-residue belongs to the binding site of cyclosporin A and is the homologous of Trp 121 in CyPA, while Trp104 residue belongs to the hydrophobic pocket. In the present work, we studied the dynamics of Trp residue(s) of cyclophilin B and of the CyPB_w128A mutant and of TNS-mutant complex. Our results showed that Trp-104 and TNS show restricted motions within their environments and that energy transfer between the two fluorophores is occurring.     

葡萄球菌核酸酶蛋白的时间分辨荧光与热力学特性

葡萄球菌核酸酶(SNase)是一种小型球状蛋白,其变体常用来研究蛋白质的折叠过程。不同于之前报道的研究方法和技术手段,采用时间相关单光子计数(TCSPC)及飞秒荧光上转换技术,结合紫外吸收谱和稳态荧光光谱,研究了SNase蛋白变体Δ+PHS和Δ+PHS+I92A的荧光动力学,以及不同温度下蛋白结构与热稳定性的关系,证明蛋白质内色氨酸残基可作为一种内源性探针对蛋白变体的结构折叠和热稳定性进行印证和研究。衰减相关光谱(DAS)表明了两种变体随温度变化的不同趋势,在此基础上进一步分析了这两种变体的结构折叠及热稳定性的差异。皮秒时间分辨发射光谱(TRES)显示色氨酸残基存在0.5 ns的连续光谱弛豫过程,而光谱移动量可作为SNase变体蛋白结构紧密程度的判断依据。飞秒上转换数据分析结果中,0.5 ps的DAS在光谱蓝端为正、红端为负,表明了色氨酸残基受到弛豫效应的影响。200 ps的寿命则说明色氨酸残基与周围猝灭基团之间存在电子转移过程。时间分辨荧光各向异性(anisotropy)的分析结果则说明了色氨酸残基在蛋白质体系内具有独立的局部运动,且其强弱与变体的热稳定性和热运动的整体效果有关。测量和分析色氨酸残基的时间分辨荧光性质为深入研究SNase蛋白的结构和功能提供了新的思路。     

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.     

Comparative Studies of Two Araceous Lectins by Steady State and Time-Resolved Fluorescence and CD Spectroscopy

Transitions in the tryptophan microenvironment and secondary structure of two monocot lectins from Sauromatum guttatum and Arisaema tortuosum under different denaturing conditions were studied by steady state and time resolved fluorescence and CD spectroscopy. The lectins exist as tetramers with a single tryptophan residue estimated per monomer, present in a polar environment. Quenching with ionic quenchers showed predominantly electropositive environment for tryptophan residues. Acrylamide had maximum quenching effect. A decrease in KI quenching due to lectin denaturation indicated redistribution of charges as a result of possible conformational change. The two values for lifetimes of tryptophanyl population (1.2–1.4 and 6.3–6.4 ns) reduced substantially on quenching or denaturation. Similarly, both the lectins showed a drastic loss of secondary structure in 5 M Gdn-HCl or 6 M Urea or at pH 2.0 and below. For the first time araceous lectins, like legume lectins are shown to bind adenine. The presence of a compact structure at alkaline pH 10.0–12.0 was observed in CD spectra.     

Dynamics ofLens culinaris agglutinin studied by red-edge excitation spectra and anisotropy measurements of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tryptophan residues

The fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate bound toLens culinaris agglutinin and of the Trp residues of the protein was investigated. Red-edge excitation spectra and steady-state anisotropy as a function of temperature indicate that the TNS is bound rigidly. Red-edge excitation spectra, steady-state anisotropy as a function of sucrose and anisotropy decay experiments performed on Trp residues fluorescence prove that the internal fluorophore presents residual motion independent of the global rotation of the protein. Fluorescence anisotropy decay allows to calculate the rotational correlation time (351 ps) of this local motion. Quenching resolved emission anisotropy with iodide gives values equal to 0.257 and 0.112 for the anisotropies of the buried and the surface Trp residues, respectively. This result indicates that the Trp residues present at the surface of the protein have important local motions compared to those embedded in the protein matrix. The results obtained from TNS and Trp residues indicate that the agglutinin has different dynamic domains.     

Quenchers Induce Wavelength Dependence on Protein Fluorescence Lifetimes

We have analysed the picosecond resolved fluorescence emission decay of horseradish peroxidase A2 and of HEW lysozyme acquired with a streak camera. Analyses of the fluorescence decay data of both proteins revealed that the dynamics of the decay is dependent on the emission wavelength. Our data strongly indicates that resonance energy transfer occurring between aromatic residues and different protein fluorescence quencher groups, and the nature of the quencher groups, are the causes of the observed wavelength dependent mean lifetime distribution. Using the global analysis data to calculate the fluorescence mean lifetime at each wavelength revealed that for lysozyme, the mean fluorescence lifetime increased with observation wavelength, whereas the opposite was the case for peroxidase. Both proteins contain strong fluorescence quencher groups located in close spatial proximity to the protein’s aromatic residues. Lysozyme contains disulfide bridges as the main fluorescence quencher whereas peroxidase contains a heme group. Both for lysozyme and horseradish peroxidase there is a clear correlation between the observed fluorescence mean lifetime of the protein at a particular emission wavelength and the respective quencher’s extinction coefficient at the respective wavelength. Furthermore, our study also reports a comparison of the analyses of the fluorescence data done with three different methods. Analyses of the fluorescence decay at 10 different fluorescence emission wavelengths revealed significant differences in both fluorescence lifetimes and the pre-exponential factor distributions. Such values differed from the values recovered from the integrated decay curves and from global analyse.     

Effect of hydrostatic pressure on the fluorescence of indole derivatives

Effects of hydrostatic pressure on the fluorescence emission of L-tryptophan, N-acetyl-L-trytophanamide and indole were investigated. An increase in pressure ranging from 1 bar to 2.4 kbar results in reversible red-shifts of the emission of the three fluorophores. The pressure-induced redshift amounts to about 170 cm^–1 at 2.4 kbar, and appears related to changes in Stokes shift of the fluorophores caused by pressure effects on the dielectric constant and/or refractive index of the medium. As the pressure range investigated here is the range commonly used in studies of protein subunit association and/or folding, these observations raise the need for caution in interpreting pressure-induced spectral shifts. The significance of these observations to pressure studies of proteins is illustrated by investigation of pressure effects on human Cu,Zn Superoxide dismutase (SOD) and azurin fromPseudomonas aeruginosa. A reversible 170 cm^–1 red-shift of the emission of SOD was observed upon pressurization to 2.4 kbar. This might be interpreted as pressure-induced conformational changes of the protein. However, further studies using SOD that had been fully unfolded by guanidine hydrochloride, and fluorescence anisotropy measurements indicated that the observed red-shift was likely due to a direct effect of pressure on the fluorescence of the single tryptophan residue of SOD. Similar pressure-induced red-shifts were also observed for the buried tryptophan residue of azurin or for azurin that had been previously denatured by guanidine hydrochloride. These observations further suggest that the effective dielectric constant of the protein matrix is affected by pressure similarly to water.     

A Study for the Cause of Ferulic Acid-Induced Quenching of Tyrosine Fluorescence and Whether it is a Reliable Marker of Intermolecular Interactions or Not

Intrinsic fluorescence of peptides and proteins is extensively used to monitor their specific interactions with several natural and synthetic molecules known to have wide-ranging beneficial or detrimental effects on health. A consequence of these interactions would be a significant decrease of the fluorescence emission intensity of Tyrosine (Tyr) and/or Tryptophan (Trp) residues in the protein due to structural rearrangements of proteic microenvironment. However fluorescence quenching can be also caused by “trivial” artefacts. In this study we examined the effect of Ferulic acid (FA) on Tyr fluorescence. FA is a natural anti-oxidant suggested to bind to and to modify the structural properties of several proteins thus altering their biological activities. Fluorescence spectroscopy experiments on Tyr and on proteins containing Tyr and no Trp like beta amyloid peptides and Insulin were performed. Our results suggest that Tyr fluorescence loss can mainly result from an inner filter effect rather than from specific interactions with FA.     

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.