UV Resonance Raman Spectroscopy Research Articles

Involvement of Histidine Residues in the pH-Dependent β-Galactoside Binding Activity of Human Galectin-1

The pH dependence of the β-galactoside binding activity of human galectin-1 (hGal-1) was investigated by fluorescence spectroscopy using lactose as a ligand. The obtained binding constant Kb was 2.94 ± 0.10 mM(-1) at pH 7.5. The Kb value decreased at acidic pH with a midpoint of transition at pH 6.0 ± 0.1. To elucidate the molecular mechanism of the pH dependence, we investigated the structures of hGal-1 and its two His mutants (H44Q and H52Q) using fluorescence, circular dichroism, UV absorption, and UV resonance Raman spectroscopy. Analysis of the spectra has shown that the pKa values of His44 and His52 are 5.7 ± 0.2 and 6.3 ± 0.1, respectively. The protonation of His52 below pH 6.3 induces a small change in secondary structure and partly reduces the galactoside binding activity. On the other hand, the protonation of His44 below pH 5.7 exerts a cation-π interaction with Trp68 and largely diminishes the galactoside binding activity. With reference to the literature X-ray structures at pH 7.0 and 5.6, protonated His52 is proposed to move slightly away from the galactoside-binding region with a partial unfolding of the β-strand containing His52. On the other hand, protonated His44 becomes unable to form a hydrogen bond with galactoside and additionally induces a reorientation and/or displacement of Trp68 through cation-π interaction, leading to a loosening of the galactoside-binding pocket. These structural changes associated with His protonation are likely to be the origin of the pH dependence of the galactoside binding activity of hGal-1.

Contribution of xylan to the brightness development and stability in the final ECF bleaching of eucalypt (Eucalyptus globulus Labill.) kraft pulp

Abstract Xylans isolated from eucalypt kraft pulps have been characterized by modern analytical methods. The pulps were partially bleached (DEOPD) and fully bleached with either a final ClO2 (DEOPDD) or an alkaline H2O2 (DEOPDP) bleaching stage. Alternatively, xylan isolated from the DEOPD pulp was treated with ClO2 or with H2O2 under the same conditions as pulps in a final bleaching stage and were further characterized. 1H nuclear magnetic resonance, size exclusion chromatography, UV-vis spectroscopy in cadoxen solution, and UV-resonance Raman spectroscopy were applied as analytical methods. The final ClO2 stage generated new unsaturated moieties in xylan, whereas H2O2 was very effective in the removal of xylan-related chromophores. The role of xylan to the delay of brightness development in the final ClO2 stage was highlighted by means of a pretreatment with xylanase Pulpzyme HC before the final bleaching stage. By this approach, the final bleachability of the pulp was boosted and the brightness stability of the fully bleached pulp was improved.

Finding the “Missing Components” during the Synthesis of TS-1 Zeolite by UV Resonance Raman Spectroscopy

The catalytic performance of TS-1 zeolite greatly depends on the types of titanium species and their concentrations in the zeolite. Coupled with UV/vis spectroscopy, we present a UV resonance Raman spectroscopic investigation on the evolution of the titanium species during the crystallization of TS-1 zeolite. It is found that a small portion of Ti species leaches from the solid phase into the liquid phase. The “missing” Ti species in addition to the framework Ti species during the assembly process of TS-1 is identified as isolated “TiO6” species, due to the resonance Raman effect excited at 266 nm. The formation mechanisms of framework Ti species in TS-1 and the relation with the isolated “TiO6” species during the synthesis process are clarified. A synthetic strategy was designed to increase the concentration of the framework titanium by a factor of 1.5 for the actual synthesis of TS-1 zeolite.

Heterogeneous Cationic Liposomes Modified with 3β-{N-[(N′,N′-Dimethylamino)ethyl]carbamoyl}cholesterol Can Induce Partial Conformational Changes in Messenger RNA and Regulate Translation in an Escherichia coli Cell-Free Translation System

The effect of cationic liposomes (CLs) on messenger RNA(mRNA) conformation and translation was studied, focusing on membrane heterogeneity. CLs, composed of 1,2-dioleoyl-sn-glycerol-3-phosphocholine/1,2-dioleoyl-3-timethylammonium propane (DOPC/DOTAP) and DOPC/3β-{N-[(N',N'-dimethylamino)ethyl]carbamoyl}cholesterol (DOPC/DC-Ch), inhibited mRNA translation in an Escherichia coli cell-free translation system. Analysis of the membrane fluidity and polarity indicated a heterogeneous DOPC/DC-Ch (70/30) membrane, while other CLs exhibited homogeneous disordered membranes. mRNA adsorbed onto DOPC/DC-Ch liposomes showed translational activity, while DOPC/DOTAP liposomes inhibited mRNA translation in proportion to its adsorption onto membranes. Dehydration of DOPC/DOTAP (70/30) and DOPC/DC-Ch (70/30) was observed in the presence of mRNA but not in the case of zwitterionic DOPC liposomes, indicating that mRNA binds in regions between the phosphate [-PO(2)(-)-] and carbonyl [-C=O-] moieties of lipids. UV resonance Raman spectroscopy suggests that adenine, cytosine, and guanine interact with DOPC/DOTAP (70/30) and DOPC/DC-Ch (70/30) but not with DOPC. Circular dichroism indicates that DOPC/DOTAP (70/30) extensively denatured the mRNA. In contrast, heterogeneous DOPC/DC-Ch (70/30) induced partial conformational changes but maintained the translational activity of mRNA.

The challenge of applying Raman spectroscopy to monitor recombinant antibody production

UV resonance Raman (UVRR) spectroscopy combined with chemometric techniques was investigated as a physiochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) cells. Due to the enhanced selectivity of the UVRR, spectral variations arising from protein, small molecule substrates, and nucleic acid medium components could be measured simultaneously and we have successfully determined antibody titre. Medium samples were taken during culture of three CHO cell lines: two antibody-producing cell lines and a non-producing cell line, and analysed by UVRR spectroscopy using an excitation laser of 244 nm. Principal component analysis (PCA) was applied to the spectral sets and showed a linear trend over time for the antibody-producing cell lines that was not observed in the non-producing cell line. Partial least squares regression (PLSR) was used to predict antibody titres, glucose utilization and lactate accumulation, and compared very favourably with gold standard data acquired with the much slower techniques of ELISA and liquid chromatography. Further analysis of the UVRR spectral sets using two-dimensional correlation moving windows also revealed that spectral variations due to protein and nucleic acid concentrations in the medium during cell culture varied between each of the three cell lines investigated.

3P092 インドールアミン2,3ジオキシゲナーゼの基質トリプトファンの検出 : 紫外共鳴ラマン分光法(02.ヘム蛋白質,ポスター,日本生物物理学会年会第51回(2013年度))

3P092 インドールアミン2,3ジオキシゲナーゼの基質トリプトファンの検出 : 紫外共鳴ラマン分光法(02.ヘム蛋白質,ポスター,日本生物物理学会年会第51回(2013年度))

Modulating Amyloid Aggregation by Incorporation of Fluorinated Phenylalanine Derivatives in the Central Hydrophobic Cluster of Aβ10-35

Aromatic amino acids have been suspected to drive the aggregation mechanism in amyloidogenic peptides by virtue of their hydrophobicity and aromaticity. Alzheimer's disease in particular is associated with the aggregation of a 39-42 residue polypeptide (Amyloid β, Aβ) and its subsequent deposition into amyloid plaques. The aromatic phenylalanine residues (Phe19 and Phe20) in the central hydrophobic cluster (CHC) of the peptide have been suspected to play a significant role in such aggregation. In the present study the amyloid β 10-35 (Aβ10-35) fragment has been used as a model system, where the CHC has been perturbed through the introduction of non-natural amino acids. We have prepared variants where Phe19 and Phe20 have been systematically replaced by pentafluorophenylalanine (F5-Phe) and their affect on amyloid aggregation and kinetics has been studied by biophysical techniques including circular dichroism (CD), fluorescence, UV resonance Raman (UVRR) spectroscopy and chemometrics. While Phe→F5-Phe mutations have been found to enhance conformational stability in Aβ16-22 (Senguen et al. Mol. BioSyst., 2011, 7, p486) by virtue of enhanced hydrophobicity, unfavorable steric interactions brought about by such substitution can also lead to destabilization in some systems (Cornilescu et al., Prot.Sc., 2007, 16, p14). Our experimental studies show that aggregation in both the wild type (wT) Aβ10-35 and the Phe 19→F5-Phe mutant proceeds via a two step conformational transition pathway (wTAβ10-35: T1= 9.18 ±1.4 hrs, T2= 9.79 ±0.44 days, F5-Phe19: T1= 10.72 ± 2.1 hrs, T2= 7.6 ± 0.47 days). However the Phe 20→F5-Phe mutants fail to aggregate into β-sheets as confirmed by both CD and fluorescence studies. Deep UVRR studies have also been performed to probe the fibrillation process and monitor the structural changes brought about by the F5-Phe substitution and their effect on amyloid aggregation.

Potentiometric chemical sensors from lignin–poly(propylene oxide) copolymers doped by carbon nanotubes

Hardwood and softwood lignins obtained from industrial sulphite and kraft and laboratory oxygen-organosolv pulping processes were employed in co-polymerization with tolylene 2,4-diisocyanate terminated poly(propylene glycol). The obtained lignin-based polyurethanes were doped with 0.72 w/w% of multiwall carbon nanotubes (MWCNTs) with the aim of increasing their electrical conductivity to the levels suitable for sensor applications. Effects of the polymer doping with MWCNTs were assessed using electrical impedance (EIS) and UV-Resonance Raman (UV-RR) spectroscopy. Potentiometric sensors were prepared by drop casting of liquid polymer on the surface of carbon glass or platinum electrodes. Lignin-based sensors displayed a very low or no sensitivity to all alkali, alkali-earth and transition metal cations ions except Cr(VI) at pH 2. Response to Cr(VI) values of 39, 50 and 53 mV pX(-1) for the sensors based on kraft, organosolv and lignosulphonate lignins, respectively, were observed. Redox sensitivity values close to the theoretical values of 20 and 21 mV pX(-1) for organosolv and lignosulphonate based sensors respectively were detected in the Cr(III)/Cr(VI) solutions while a very low response was observed in the solutions containing Fe(CN)(6)(3-/4-). Conducting composite lignin-based polyurethanes doped with MWCNTs were suggested as being promising materials for Cr(VI)-sensitive potentiometric sensors.

146 Amyloid fibril polymorphism probed by advanced vibrational spectroscopy

Amyloid fibrils are associated with many neurodegenerative diseases. All known amyloids including pathogenic and nonpathogenic forms display functional and structural heterogeneity (polymorphism) which determines the level of their toxicity. Despite a significant biological and biomedical importance, the nature of the amyloid fibril polymorphism remains elusive. We utilized for the first time three most advanced vibrational techniques to probe the core, the surface, and supramolecular chirality of fibril polymorphs. A new type of folding, aggregation phenomenon, spontaneous refolding from one polymorph to another, was discovered (Kurouski, Lauro et al., 2010). Hydrogen–deuterium exchange deep UV resonance Raman spectroscopy (Oladepo, Xiong et al., 2012) combined with advanced statistical analysis (Shashilov & Lednev, 2010) allowed for structural characterization of the highly ordered cross-β core of amyloid fibrils. We reported several examples showing significant variations in the core structure for fibril polymorphs. Amyloid fibrils are generally composed of several protofibrils and may adopt variable morphologies, such as twisted ribbons or flat-like sheets. We discovered the existence of another level of amyloid polymorphism, namely, that associated with fibril supramolecular chirality. Two chiral polymorphs of insulin, which can be controllably grown by means of small pH variations, exhibit opposite signs of vibrational circular dichroism (VCD) spectra (Kurouski, Dukor et al. 2012). VCD supramolecular chirality is correlated not only by the apparent fibril handedness but also by the sense of supramolecular chirality from a deeper level of chiral organization at the protofilament level of fibril structure. A small pH change initiates spontaneous transformation of insulin fibrils from one polymorph to another. As a result, fibril supramolecular chirality overturns both accompanying morphological and structural changes (Kurouski, Dukor et al. 2012). No conventional methods could probe the fibril surface despite its significant role in the biological activity. We utilized tip-enhanced Raman spectroscopy (TERS) to characterize the surface structure of an individual fibril due to a high depth and lateral spatial resolution of the method in the nanometer range (Kurouski, Deckert-Gaudig et al. 2012). It was found that the surface is strongly heterogeneous and consists of clusters with various protein conformations and amino acid composition.

Deep UV resonance Raman spectroscopic study of CnF2n+2molecules: the excitation of C-C σ bond

The σ–σ* transition of C–C bond in CnF2n+2 molecules was studied by deep UV resonance Raman spectroscopy. With the C–C σ bond selectively excited by the deep UV laser at 177.3 nm, the resonance Raman spectra of CnF2n+2 molecules were obtained on our home-assembled deep UV Raman spectrograph. The Raman bands at 1299, 1380 and 2586 cm−1 due to the C–C skeletal stretching modes are evidently enhanced owing to the resonance Raman effect. Based on the resonance Raman spectra and theoretical calculation results, it is proposed that the electronic geometry of CnF2n+2 molecules at the σσ* excited state is displaced along the directions perpendicular and parallel to the C–C skeleton, and the excited C–C bond is not dissociative due to the delocalization of the excited electron in σ* orbital. Copyright © 2012 John Wiley & Sons, Ltd.

Effect of urea on cellulose degradation under conditions of alkaline pulping

The effect of urea on the cellulose degradation under conditions of alkaline pulping has been studied using purified cellulose powder. The increased cellulose yield in the presence of urea was assigned essentially to the carbamation reactions which were confirmed by elemental analysis, UV-resonance Raman and solid-state 13C nuclear magnetic resonance spectroscopy. The stabilizing effect of urea on the cellulose peeling reactions during heating up period of pulping process was suggested based on kinetic studies and additionally confirmed in model reactions using cellobiose. The reaction products formed in alkaline urea solutions were analysed by tandem electrospray ionization mass spectrometry and the occurrence of Maillard type reactions between reducing end groups of cellobiose and urea were evidenced. Both Maillard type reactions and carbamation of reducing end groups were proposed to be a part of cellulose protection mechanism against peeling under the conditions of alkaline pulping.

A Thorough Investigation of the Active Titanium Species in TS‐1 Zeolite by In Situ UV Resonance Raman Spectroscopy

A thorough investigation of the active titanium species in TS-1 zeolite was conducted by in situ UV resonance Raman spectroscopy combined with UV/Vis diffuse reflectance spectroscopy, DFT calculations, and epoxidation experiments. A new titanium species was identified with a characteristic Raman band at 695 cm(-1) when excited at the 266 nm laser line. It is shown that the newly found titanium species is active in the epoxidation reactions in addition to the tetrahedrally coordinated titanium species. However, the acidity of the new titanium species could catalyze the ring-opening reactions of the epoxy products. It results in a lower selectivity toward the epoxy products relative to that of the tetrahedrally coordinated titanium species. The side reaction can be suppressed by the addition of a weak basic reagent.

N-Terminal Truncation Does Not Affect the Location of a Conserved Tryptophan in the BLUF Domain of AppA from Rhodobacter sphaeroides

The flavin-binding BLUF domains are a class of blue-light receptors, and AppA is a representative of this family. Although the crystal and solution structures of several BLUF domains have already been obtained, there is a key uncertainty regarding the position of a functionally important tryptophan (Trp104 in AppA). In the first crystal structure of an N-terminally truncated BLUF domain of AppA133 (residues 17-133), Trp104 was found in close proximity to flavin (Trp(in)), whereas in a subsequent structure with an intact N-terminus AppA126 (residues 1-126), Trp104 was exposed to the solvent (Trp(out)). A recent study compared spectroscopic properties of AppA126 and AppA133 and claimed that the Trp(in) conformation is an artifact of N-terminal truncation in AppA133. In this study, we compared the flavin vibrational spectra of AppA126 and AppA133 by using near-infrared excited Raman spectroscopy. In addition, the conformations as well as the environments of Trp104 were directly monitored by ultraviolet resonance Raman spectroscopy. These studies demonstrate that the N-terminal truncation does not induce the conformational switch between Trp(in) and Trp(out).

UV resonance Raman spectroscopy monitors polyglutamine backbone and side chain hydrogen bonding and fibrillization.

We utilize 198 and 204 nm excited UV resonance Raman spectroscopy (UVRR) and circular dichroism spectroscopy (CD) to monitor the backbone conformation and the Gln side chain hydrogen bonding (HB) of a short, mainly polyGln peptide with a D(2)Q(10)K(2) sequence (Q10). We measured the UVRR spectra of valeramide to determine the dependence of the primary amide vibrations on amide HB. We observe that a nondisaggregated Q10 (NDQ10) solution (prepared by directly dissolving the original synthesized peptide in pure water) exists in a β-sheet conformation, where the Gln side chains form hydrogen bonds to either the backbone or other Gln side chains. At 60 °C, these solutions readily form amyloid fibrils. We used the polyGln disaggregation protocol of Wetzel et al. [Wetzel, R., et al. (2006) Methods Enzymol.413, 34-74] to dissolve the Q10 β-sheet aggregates. We observe that the disaggregated Q10 (DQ10) solutions adopt PPII-like and 2.5(1)-helix conformations where the Gln side chains form hydrogen bonds with water. In contrast, these samples do not form fibrils. The NDQ10 β-sheet solution structure is essentially identical to that found in the NDQ10 solid formed upon evaporation of the solution. The DQ10 PPII and 2.5(1)-helix solution structure is essentially identical to that in the DQ10 solid. Although the NDQ10 solution readily forms fibrils when heated, the DQ10 solution does not form fibrils unless seeded with the NDQ10 solution. This result demonstrates very high activation barriers between these solution conformations. The NDQ10 fibril secondary structure is essentially identical to that of the NDQ10 solution, except that the NDQ10 fibril backbone conformational distribution is narrower than in the dissolved species. The NDQ10 fibril Gln side chain geometry is more constrained than when NDQ10 is in solution. The NDQ10 fibril structure is identical to that of the DQ10 fibril seeded by the NDQ10 solution.

Initial Excited-State Structural Dynamics of Thymine Derivatives

Thymine is one of the pyrimidine nucleobases found in DNA. Upon absorption of UV light, thymine forms a number of photoproducts, including the cyclobutyl photodimer, the pyrimidine pyrimidinone [6-4] photoproduct and the photohydrate. Here, we use UV resonance Raman spectroscopy to measure the initial excited-state structural dynamics of the N(1)-substituted thymine derivatives N(1)-methylthymine, thymidine, and thymidine 5'-monophosphate in an effort to understand the role of the N1 substituent in determining the excited-state structural dynamics and the subsequent photochemistry. The UV resonance Raman spectrum of thymidine and thymidine 5'-monophosphate are similar to that of thymine, suggesting that large masses at N(1) effectively isolate the vibrations of the nucleobase. However, the UV resonance Raman spectrum of N(1)-methylthymine is significantly different, suggesting that the methyl group couples into the thymine ring vibrations. The resulting resonance Raman intensities and absorption spectra are self-consistently simulated with a time-dependent expression to quantitatively extract the initial excited-state slopes, homogeneous and inhomogeneous linewidths, and electronic parameters. These results are discussed in the context of the known photochemistry of thymine and its derivatives.

UV resonance Raman study of TrpZip2 and related peptides: π-π interactions of tryptophan.

Aromatic interactions are important stabilizing forces in proteins but are difficult to detect in the absence of high-resolution structures. Ultraviolet resonance Raman spectroscopy is used to probe the vibrational signatures of aromatic interactions in TrpZip2, a synthetic β-hairpin peptide that is stabilized by edge-to-face and face-to-face tryptophan π-π interactions. The vibrational markers of isolated edge-to-face π-π interactions are investigated in the related β-hairpin peptide W2W11. The bands that comprise the Fermi doublet exhibit systematic shifts in position and intensity for TrpZip2 and W2W11 relative to the model peptide, W2W9, which does not form aromatic interactions. Additionally, hypochromism of the Bb absorption band of tryptophan in TrpZip2 leads to a decrease in the relative Raman cross-sections of Bb-coupled Raman bands. These results reveal spectral markers for stabilizing tryptophan π-π interactions and indicate that ultraviolet resonance Raman may be an important tool for the characterization of these biological forces.

Impact of ion binding on poly-L-lysine (un)folding energy landscape and kinetics.

We utilize T-jump UV resonance Raman spectroscopy (UVRR) to study the impact of ion binding on the equilibrium energy landscape and on (un)folding kinetics of poly-L-lysine (PLL). We observe that the relaxation rates of the folded conformations (including π-helix (bulge), pure α-helix, and turns) of PLL are slower than those of short alanine-based peptides. The PLL pure α-helix folding time is similar to that of short alanine-based peptides. We for the first time have directly observed that turn conformations are α-helix and π-helix (bulge) unfolding intermediates. ClO(4)(-) binding to the Lys side chain -NH(3)(+) groups and the peptide backbone slows the α-helix unfolding rate compared to that in pure water, but little impacts the folding rate, resulting in an increased α-helix stability. ClO(4)(-) binding significantly increases the PLL unfolding activation barrier but little impacts the folding barrier. Thus, the PLL folding coordinate(s) differs from the unfolding coordinate(s). The-π helix (bulge) unfolding and folding coordinates do not directly go through the α-helix energy well. Our results clearly demonstrate that PLL (un)folding is not a two-state process.

Disulfide Bridges Remain Intact while Native Insulin Converts into Amyloid Fibrils

Amyloid fibrils are β-sheet-rich protein aggregates commonly found in the organs and tissues of patients with various amyloid-associated diseases. Understanding the structural organization of amyloid fibrils can be beneficial for the search of drugs to successfully treat diseases associated with protein misfolding. The structure of insulin fibrils was characterized by deep ultraviolet resonance Raman (DUVRR) and Nuclear Magnetic Resonance (NMR) spectroscopy combined with hydrogen-deuterium exchange. The compositions of the fibril core and unordered parts were determined at single amino acid residue resolution. All three disulfide bonds of native insulin remained intact during the aggregation process, withstanding scrambling. Three out of four tyrosine residues were packed into the fibril core, and another aromatic amino acid, phenylalanine, was located in the unordered parts of insulin fibrils. In addition, using all-atom MD simulations, the disulfide bonds were confirmed to remain intact in the insulin dimer, which mimics the fibrillar form of insulin.

Fibrillation Mechanism of a Model Intrinsically Disordered Protein Revealed by 2D Correlation Deep UV Resonance Raman Spectroscopy

Understanding of numerous biological functions of intrinsically disordered proteins (IDPs) is of significant interest to modern life science research. A large variety of serious debilitating diseases are associated with the malfunction of IDPs including neurodegenerative disorders and systemic amyloidosis. Here we report on the molecular mechanism of amyloid fibrillation of a model IDP (YE8) using 2D correlation deep UV resonance Raman spectroscopy. YE8 is a genetically engineered polypeptide, which is completely unordered at neutral pH yet exhibits all properties of a fibrillogenic protein at low pH. The very first step of the fibrillation process involves structural rearrangements of YE8 at the global structure level without the detectable appearance of secondary structural elements. The formation of β-sheet species follows the global structural changes and proceeds via the simultaneous formation of turns and β-strands. The kinetic mechanism revealed is an important new contribution to understanding of the general fibrillation mechanism proposed for IDP.

PH-Dependent Complexation of Histamine H1 Receptor Antagonists and Human Serum Albumin Studied by UV Resonance Raman Spectroscopy

UV resonance Raman spectroscopy was used to characterize the binding of three first-generation histamine H(1) receptor antagonists-tripelennamine (TRP), mepyramine (MEP), and brompheniramine (BPA)-to human serum albumin (HSA) at pH 7.2 and pH 9.0. Binding constants differ at these pH values, which can be ascribed to the different extent of protonation of the ethylamino side chain of the ligands. We have recently shown [Tardioli et al. J. Raman Spectrosc. 2011, 42, 1016-1024] that for the solution conformation of TRP and MEP the side chain plays an important role by allowing an internal hydrogen bond with the aminopyridine nitrogen in TRP and MEP. Results presented in this paper suggest that the existence of such molecular structures has serious biological significance on the binding affinity of those ligands to HSA. At pH 7.2, only the stretched conformers of protonated TRP and MEP bind in HSA binding site I. Using UV absorption data, we derived binding constants for the neutral and protonated forms of TRP to HSA. The neutral species seems to be conjugated to a positive group of the protein, affecting both the tryptophan W214 and some of the tyrosine (Y) vibrations. BPA, for which the structure with an intramolecular hydrogen bonded side chain is not possible, is H bound to the indole ring nitrogen of W214, of which the side chain rotates over a certain angle to accommodate the drug in site I. We propose that the protonated BPA is also bound in site I, where the Y150 residue stabilizes the presence of this compound in the binding pocket. No spectroscopic evidence was found for conformational changes of the protein affecting the spectroscopic properties of W and Y in this pH range.