Trp scanning analysis of Tet repressor reveals conformational changes associated with operator and anhydrotetracycline binding.

We analysed the conformational states of free, tet operator-bound and anhydrotetracycline-bound Tet repressor employing a Trp-scanning approach. The two wild-type Trp residues in Tet repressor were replaced by Tyr or Phe and single Trp residues were introduced at each of the positions 162-173, representing part of an unstructured loop and the N-terminal six residues of alpha-helix 9. All mutants retained in vivo inducibility, but anhydrotetracycline-binding constants were decreased up to 7.5-fold when Trp was in positions 169, 170 and 173. Helical positions (168-173) differed from those in the loop (162-167) in terms of their fluorescence emission maxima, quenching rate constants with acrylamide and anisotropies in the free and tet operator-complexed proteins. Trp fluorescence emission decreased drastically upon atc binding, mainly due to energy transfer. For all proteins, either free, tet operator bound or anhydrtetracycline-bound, mean fluorescence lifetimes were determined to derive quenching rate constants. Solvent-accessible surfaces of the respective Trp side chains were calculated and compared with the quenching rate constants in the anhydrotetracycline-bound complexes. The results support a model, in which residues in the loop become more exposed, whereas residues in alpha-helix 9 become more buried upon the induction of TetR by anhydrotetracycline.     

Fluorescence contributions of the individual Trp residues in goat alpha-lactalbumin

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues. In order to obtain information on the fluorescence contribution of the individual Trp residues in native GLA, we recorded the fluorescence spectra of four GLA mutants, W26F, W60F, W104F, and W118F, in each of which a single Trp residue was replaced with phenylalanine (Phe). Comparison of the fluorescence spectra of the four mutants with that of wild-type GLA indicated that, in native GLA, three Trp residues (Trp60, Trp104, and Trp118) are strongly quenched and account for the partial indirect quenching of Trp26. As a consequence, the fluorescence of wild-type GLA and of the mutants W60F, W104F, and W118F mainly results from Trp26. An inspection of the crystal structure indicated that, in addition to the disulfide bonds that are in direct contact with the indole groups of Trp60 and Trp118, backbone peptide bonds that are in direct contact with the indole groups of Trp60, Trp104, and Trp118, contribute to the direct quenching effects. Interestingly, the lack of direct quenching of Trp26 explains why the cleavage of disulfide bonds by UV light is mediated more by the highly fluorescent Trp26 than by the less fluorescent Trp104 and Trp118.     

Structural studies of mitochondrial coupling factor 1 using tyrosine fluorescence

The absorbance and fluorescence spectral properties of mitochondrial F1-ATPase confirm that this protein does not contain tryptophan residues and therefore its fluorescence is due to tyrosines. The 36% increase in the fluorescence and the almost 100% increase in quantum yield upon denaturation of the protein suggest that a considerable number of tyrosyl residues have a very low quantum yield in the native enzyme. Quenching experiments using iodide indicate that all of the fluorophores are quenched and also all of them with the same quenching constant. These observations are interpreted as confirmatory of what has been found with several other proteins whose fluorescence originates from tyrosyl residues, where the buried tyrosines fluoresce with a much lower quantum yield than those which are exposed. ATP added to F1 previously depleted of loosely bound nucleotides changes the quenching constant of iodide and the quantum yield and this is interpreted to be due to a conformational change induced by the binding of the nucleotide to the enzyme. Addition of 2-mercaptoethanol decreases, although slightly, the polarization of the fluorescence. However, SDS addition gives a much bigger decrease. Hence disulphide bridges are less important for the tertiary structure of the protein than hydrophobic interactions, hydrogen bonding or other forces. Nevertheless the conformational change induced by reduction of disulphide bridges is detected in iodide quenching experiments and the change of the quantum yield of the enzyme.     

Mechanism of the highly efficient quenching of tryptophan fluorescence in human gammaD-crystallin

Quenching of the fluorescence of buried tryptophans (Trps) is an important reporter of protein conformation. Human gammaD-crystallin (HgammaD-Crys) is a very stable eye lens protein that must remain soluble and folded throughout the human lifetime. Aggregation of non-native or covalently damaged HgammaD-Crys is associated with the prevalent eye disease mature-onset cataract. HgammaD-Crys has two homologous beta-sheet domains, each containing a pair of highly conserved buried tryptophans. The overall fluorescence of the Trps is quenched in the native state despite the absence of the metal ligands or cofactors. We report the results of detailed quantitative measurements of the fluorescence emission spectra and the quantum yields of numerous site-directed mutants of HgammaD-Crys. From fluorescence of triple Trp to Phe mutants, the homologous pair Trp68 and Trp156 were found to be extremely quenched, with quantum yields close to 0.01. The homologous pair Trp42 and Trp130 were moderately fluorescent, with quantum yields of 0.13 and 0.17, respectively. In an attempt to identify quenching and/or electrostatically perturbing residues, a set of 17 candidate amino acids around Trp68 and Trp156 were substituted with neutral or hydrophobic residues. None of these mutants showed significant changes in the fluorescence intensity compared to their own background. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations with the four different excited Trps as electron donors strongly indicate that electron transfer rates to the amide backbone of Trp68 and Trp156 are extremely fast relative to those for Trp42 and Trp130. This is in agreement with the quantum yields measured experimentally and consistent with the absence of a quenching side chain. Efficient electron transfer to the backbone is possible for Trp68 and Trp156 because of the net favorable location of several charged residues and the orientation of nearby waters, which collectively stabilize electron transfer electrostatically. The fluorescence emission spectra of single and double Trp to Phe mutants provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain. The backbone conformation of tryptophans in HgammaD-Crys may have evolved in part to enable the lens to become a very effective UV filter, while the efficient quenching provides an in situ mechanism to protect the tryptophans of the crystallins from photochemical degradation.     

N5-(L-1-carboxyethyl)-L-ornithine synthase: physical and spectral characterization of the enzyme and its unusual low pKa fluorescent tyrosine residues.

N5-(L-1-carboxyethyl)-L-ornithine synthase [E.C. 1.5.1.24] (CEOS) from Lactococcus lactis has been cloned, expressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods. The NADPH-dependent enzyme is a homotetramer (Mr approximately equal to 140,000) and in the native state is stabilized by noncovalent interactions between the monomers. The far-ultraviolet circular dichroism spectrum shows that the folding pattern of the enzyme is typical of the alpha,beta family of proteins. CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, and the fluorescence spectrum of the protein shows emission from both Trp and Tyr residues. Relative to N-acetyltyrosinamide, the Tyr quantum yield of the native enzyme is about 0.5. All 19 Tyr residues are titratable and, of these, two exhibit the uncommonly low pKa of approximately 8.5, 11 have pKa approximately 10.75, and the remaining six titrate with pKa approximately 11.3. The two residues with pKa approximately 8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicating Tyr-Tyr energy transfer. In the presence of NADPH, Tyr fluorescence is reduced by 40%, and Trp fluorescence is quenched completely. The latter result suggests that the single Trp residue is either at the active site, or in proximity to the sequence GSGNVA, that constitutes the beta alphabeta fold of the nucleotide-binding domain. Chymotrypsin specifically cleaves native CEOS after Phe255. Although inactivated by this single-site cleavage of the subunit, the enzyme retains the capacity to bind NADPH and tetramer stability is maintained. Possible roles in catalysis for the chymotrypsin sensitive loop and for the low pKa Tyr residues are discussed.     

Probing the renin active site by collisional quenching of endogenous fluorescence

The structural and enzymatic aspects of renin are of great interest in hypertension research. In this paper, we examine the solution accessibility of the three tryptophan (Trp) residues of mouse submaxillary gland renin by solute collisional fluorescence quenching. Our studies indicate that there are two "classes" of Trp residues in renin: class I, a class of Trp residues which are at or near the surface of renin and fully accessible to the fluorescence quencher iodide; and class II, a class of Trp residues which are, for practical experimental conditions, totally inaccessible to the aqueous solution. The former class contains 2 Trp residues, while only a single Trp is identified in the latter class. The presence of a tetradecapeptide substrate or a nonhydrolyzable substrate analogue (peptide H-77) lowers the accessibility of iodide to the class I Trp residues. These data indicate that the class I Trp residues are at or near the peptide-binding site of renin. In addition, the finding that the class I Trp residues are quantitatively quenched more efficiently than the Trp model compound indole suggests that the environment of the class I tryptophans may be positively charged, and thus have a higher "local" concentration of iodide. These data, taken together with the available sequence and computer-generated three-dimensional structure of renin, permit us to speculate that the class I Trp residues are Trp-39 and Trp-300. This solution study of renin structure is discussed in light of the known information about renin catalysis and physiology.     

Conformational dynamics of DnaB helicase upon DNA and nucleotide binding: analysis by intrinsic tryptophan fluorescence quenching

DnaB helicase of E. coli unwinds duplex DNA in the replication fork using the energy of ATP hydrolysis. We have analyzed structural and conformational changes in the DnaB protein in various nucleotides and DNA bound intermediate states by fluorescence quenching analysis of intrinsic fluorescence of native tryptophan (Trp) residues in DnaB. Fluorescence quenching analysis indicated that Trp48 in domain alpha is in a hydrophobic environment and resistant to fluorescence quenchers such as potassium iodide (KI). In domain beta, Trp294 was found to be in a partially hydrophobic environment, whereas Trp456 in domain gamma appeared to be in the least hydrophobic environment. Binding of oligonucleotides to DnaB helicase resulted in a significant attenuation of the fluorescence quenching profile, indicating a change in conformation. ATPgammaS or ATP binding appeared to lead to a conformation in which Trp residues had a higher degree of solvent exposure and fluorescence quenching. However, the most dramatic increase of Trp fluorescence quenching was observed with ADP binding with a possible conformational relaxation. Site-specific Trp --> Cys mutants of DnaB helicase demonstrated that conformational change upon ADP binding could be attributed exclusively to a conformational transition in the alpha domain leading to an increase in the solvent exposure of Trp48. However, formation of DnaB.ATPgammaS.DNA ternary complex led to a conformation with a fluorescence quenching profile similar to that observed with DnaB alone. The DnaB.ADP.DNA ternary complex produced a quenching curve similar to that of DnaB.ADP complex pointing to a change in conformation due to ATP hydrolysis. There are at least four identifiable structural/conformational states of DnaB helicase that are likely important in the helicase activity. The noncatalytic alpha domain in the N-terminus appeared to undergo the most significant conformational changes during nucleotide binding and hydrolysis. This is the first reported elucidation of the putative role of domain alpha, which is essential for DNA helicase action. We have correlated these results with partial structural models of alpha, beta, and gamma domains     

Energy transfer from tryptophan residues of proteins to 8-anilinonaphthalene-1-sulfonate

The binding of the apolar fluorescent dye 8-anilinonaphthalene-1-sulfonate (ANS) to bovine serum albumin (BSA), phospholipase A₂ (PLA₂), ovalbumin, lysozyme, cobrotoxin and N-acetyltryptophanamide was used to assess the factors affecting the efficiency of energy transfer from Trp residues to the ANS molecule. We found that the efficiency of energy transfer from Trp residues to ANS was associated with the ability of proteins to enhance the ANS fluorescence. At the same molar concentration of protein, BSA enhanced ANS fluorescence most among these proteins; its Trp fluorescence was drastically quenched by the addition of ANS. Fluorescence enhancement of ANS in PLA₂-ANS complex increased upon addition of Ca²⁺ or change of the buffer to acidicpH, resulting in a higher efficiency of energy transfer from Trp residues to ANS. There was limited ANS fluorescence enhancement with ovalbumin, lysozyme, cobrotoxin, and N-acetyltryptophanamide and a less efficient quenching in Trp fluorescence. The capabilities of proteins for binding with ANS correlated with the decrease in their Trp fluorescence being quenching by ANS. However, the microenvironment surrounding Trp residues of proteins did not affect the energy transfer. Based on these results, the factors that affected the energy transfer from Trp residues to ANS are discussed.     

Assessment of the role of tryptophan residues in phospholipase A2 by fluorescence quenching

Tryptophan (Trp) fluorescence of two phospholipases A2 (PLA2) from Naja naja atra and Naja nigricollis snake venoms was quenched by acrylamide and iodide. Trp residues in N. naja atra PLA2 were equally accessible to acrylamide and iodide. Iodide quenching studies indicate that there are two classes of Trp fluorophores in N. nigricollis CMS-9. The accessible class consists of Trp-18 and Trp-19. Removal of the N-terminal octapeptide caused a perturbation of the micro-environment of the Trp residues in the PLA2 enzymes. The presence of a substrate lowers the susceptibility of the Trp residues to iodide quenching in N. naja atra PLA2, suggesting that all three Trp residues are at the substrate binding site, but in N. nigricollis CMS-9 Trp-18 and Trp-19 are related to substrate binding.     

Probing folding and fluorescence quenching in human gammaD crystallin Greek key domains using triple tryptophan mutant proteins

Human gammaD crystallin (HgammaD-Crys), a major component of the human eye lens, is a 173-residue, primarily beta-sheet protein, associated with juvenile and mature-onset cataracts. HgammaD-Crys has four tryptophans, with two in each of the homologous Greek key domains, which are conserved throughout the gamma-crystallin family. HgammaD-Crys exhibits native-state fluorescence quenching, despite the absence of ligands or cofactors. The tryptophan absorption and fluorescence quenching may influence the lens response to ultraviolet light or the protection of the retina from ambient ultraviolet damage. To provide fluorescence reporters for each quadrant of the protein, triple mutants, each containing three tryptophan-to-phenylalanine substitutions and one native tryptophan, have been constructed and expressed. Trp 42-only and Trp 130-only exhibited fluorescence quenching between the native and denatured states typical of globular proteins, whereas Trp 68-only and Trp 156-only retained the anomalous quenching pattern of wild-type HgammaD-Crys. The three-dimensional structure of HgammaD-Crys shows Tyr/Tyr/His aromatic cages surrounding Trp 68 and Trp 156 that may be the source of the native-state quenching. During equilibrium refolding/unfolding at 37 degrees C, the tryptophan fluorescence signals indicated that domain I (W42-only and W68-only) unfolded at lower concentrations of GdnHCl than domain II (W130-only and W156-only). Kinetic analysis of both the unfolding and refolding of the triple-mutant tryptophan proteins identified an intermediate along the HgammaD-Crys folding pathway with domain I unfolded and domain II intact. This species is a candidate for the partially folded intermediate in the in vitro aggregation pathway of HgammaD-Crys.     

Fluorescence of native single-Trp mutants in the lactose permease from Escherichia coli: structural properties and evidence for a substrate-induced conformational change.

Six single-Trp mutants were engineered by individually reintroducing each of the native Trp residues into a functional lactose permease mutant devoid of Trp (Trp-less permease; Menezes ME, Roepe PD, Kaback HR, 1990, Proc Natl Acad Sci USA 87:1638-1642), and fluorescent properties were studied with respect to solvent accessibility, as well as alterations produced by ligand binding. The emission of Trp 33, Trp 78, Trp 171, and Trp 233 is strongly quenched by both acrylamide and iodide, whereas Trp 151 and Trp 10 display a decrease in fluorescence in the presence of acrylamide only and no quenching by iodide. Of the six single-Trp mutants, only Trp 33 exhibits a significant change in fluorescence (ca. 30% enhancement) in the presence of the substrate analog beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). This effect was further characterized by site-directed fluorescent studies with purified single-Cys W33-->C permease labeled with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS). Titration of the change in the fluorescence spectrum reveals a 30% enhancement accompanied with a 5-nm blue shift in the emission maximum, and single exponential behavior with an apparent KD of 71 microM. The effect of substrate binding on the rate of MIANS labeling of single-Cys 33 permease was measured in addition to iodide and acrylamide quenching of the MIANS-labeled protein. Complete blockade of labeling is observed in the presence of TDG, as well as a 30% decrease in accessibility to iodide with no change in acrylamide quenching. Overall, the findings are consistent with the proposal (Wu J, Frillingos S, Kaback HR, 1995a, Biochemistry 34:8257-8263) that ligand binding induces a conformational change at the C-terminus of helix I such that Pro 28 and Pro 31, which are on one face, become more accessible to solvent, whereas Trp 33, which is on the opposite face, becomes less accessible to the aqueous phase. The findings regarding accessibility to collisional quenchers are also consistent with the predicted topology of the six native Trp residues in the permease.     

Relation between the secondary structure of carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and the fluorescence of the protein

We studied in this work the relation that exists between the secondary structure of the glycans of alpha(1)-acid glycoprotein and the fluorescence of the Trp residues of the protein. We calculated for that the efficiency of quenching and the radiative and non-radiative constants. Our results indicate that the glycans display a spatial structure that is modified upon asialylation. The asialylated conformation is closer to the protein matrix than the sialylated form, inducing by that a decrease in the fluorescence parameters of the Trp residues. In fact, the mean quantum yield of Trp residues in sialylated and asialylated alpha(1)-acid glycoprotein are 0.0645 and 0.0385, respectively. Analysis of the fluorescence emission of alpha(1)-acid glycoprotein as the result of two contributions (surface and hydrophobic domains) indicates that quantum yields of both classes of Trp residues are lower when the protein is in the asialylated form. Also, the mean fluorescence lifetime of Trp residues decreases from 2.285 ns in the sialylated protein to 1.948 ns in the asialylated one. The radiative rate constant k(r) of the Trp residues in the sialylated alpha(1)-acid glycoprotein is higher than that in the asialylated protein. Thus, the carbohydrate residues are closer to the Trp residues in the absence of sialic acid. The modification of the spatial conformation of the glycans upon asialylation is confirmed by the decrease of the fluorescence lifetimes of Calcofluor, a fluorophore that binds to the carbohydrate residues. Finally, thermal intensity quenching of Calcofluor bound to alpha(1)-acid glycoprotein shows that the carbohydrate residues have slower residual motions in the absence of sialic acid residues.     

A Concerted Tryptophanyl-adenylate-dependent Conformational Change inBacillus subtilisTryptophanyl-tRNA Synthetase Revealed by the Fluorescence of Trp92

A semi-conserved tryptophan residue ofBacillus subtilistryptophanyl-tRNA synthetase (TrpRS) was previously asserted to be an essential residue and directly involved in tRNA^Trpbinding and recognition. The crystal structure of theBacillus stearothermophilusTrpRS tryptophanyl-5′-adenylate complex (Trp-AMP) shows that the corresponding Trp91 is buried and in the dimer interface, contrary to the expectations of the earlier assertation. Here we examine the role of this semi-conserved tryptophan residue using fluorescence spectroscopy.B. subtilisTrpRS has a single tryptophan residue, Trp92. 4-Fluorotryptophan (4FW) is used as a non-fluorescent substrate analog, allowing characterization of Trp92 fluorescence in the 4-fluorotryptophanyl-5′-adenylate (4FW-AMP) TrpRS complex. Complexation causes the Trp92 fluorescence to become quenched by 70%. Titrations, forming this complex under irreversible conditions, show that this quenching is essentially complete after half of the sites are filled. This indicates that a substrate-dependent mechanism exists for the inter-subunit communication of conformational changes. Trp92 fluorescence is not efficiently quenched by small solutes in either the apo- or complexed form. From this we conclude that this tryptophan residue is not solvent exposed and that binding of the Trp92 to tRNA^Trpis unlikely.Time-resolved fluorescence indicates conformational heterogeneity ofB. subtilisTrp92 with the fluorescence decay being best described by three discrete exponential decay times. The decay-associated spectra (DAS) of the apo- and complexed- TrpRS show large variations of the concentration of individual fluorescence decay components. Based on recent correlations of these data with changes in the local secondary structure of the backbone containing the fluorescent tryptophan residue, we conclude that changes observed in Trp92 time-resolved fluorescence originate primarily from large perturbations of its local secondary structure.The quenching of Trp92 in the 4FW-AMP complex is best explained by the crystal structure conformation, in which the tryptophan residue is found in an α-helix. The amino acid residue cysteine is observed clearly within the quenching radius (3.6 Å) of the conserved tryptophan residue. These tryptophan and cysteine residues are neighbors, one helical turn apart. If this local α-helix was disrupted in the apo-TrpRS, this disruption would concomitantly relieve the putative cysteine quenching by separating the two residues. Hence we propose a substrate-dependent local helix-coil transition to explain both the observed time-resolved and steady-state fluorescence of Trp92. A mechanism can be further inferred for the inter-subunit communication involving the substrate ligand Asp132 and a small α-helix bridging the substrate tryptophan residue and the conserved tryptophan residue of the opposite subunit. This putative mechanism is also consistent with the observed pH dependence of TrpRS crystal growth and substrate binding. We observe that the mechanism of TrpRS has a dynamic component, and contend that conformational dynamics of aminoacyl-tRNA synthetases must be considered as part of the molecular basis for the recognition of cognate tRNA.     

Comparative fluorescence properties of bovine,goat, human and guinea pig α lactalbumin: Characterization of the environments of individual tryptophan residues in partially folded conformers

α Lactalbumin exists as a partially folded conformer (U form) at acid pH. A second partially folded conformer (H form) is formed above 60°. Comparison of the changes in tryptophan fluorescence which occur on forming U and H for the bovine, goat, human and guinea pig proteins, as well as analysis of fluorescence properties for the bovine protein and an N bromo succinimide derivative of this protein, have made it possible to determine which tryptophan residues give rise to such changes in fluorescence, and to draw a distinction between the molecular structure of the U and H forms of the protein. Trp 28 and 109 in the native state transfer their excitation energy to trp 63 whose fluorescence is quenched by a pair of vicinal disulfide bridges. This process persists in the U form of the protein, but is absent in the H conformer. Most of the change in fluorescence seen in the N ? U conversion is due to increase in yield of trp 28, while the changes in fluorescence occurring on formation of the H form are due to exposure of trp 63 and elimination of its quenching and/or excited state transfer from 28 to 109.     

The uncoupling protein from brown adipose tissue mitochondria. The environment of the tryptophan residues as revealed by quenching of the intrinsic fluorescence.

The uncoupling protein from brown adipose tissue is a member of the family of metabolite carriers of the mitochondrial inner membrane. It contains two tryptophan residues which have been characterized by fluorescence spectroscopy. Application of fluorescence-quenching-resolved spectroscopy (FQRS) allowed the determination of the emission maximum for each residue, both of which occur at 332 nm, thus suggesting that they are both located in a non-polar environment. Fluorescence quenching has demonstrated that both residues are accessible to acrylamide and inaccessible to Cs+, while only one of them is accessible to I-. When FQRS is combined with guanidinium hydrochloride denaturation, the unfolding of the regions containing each tryptophan can be monitored separately as they are transferred to the polar medium where the emission maximum appears at 359 nm, revealing also that the iodide-accessible residue is more sensitive to the denaturant. Secondary structure predictions, together with the data presented here, suggest that the iodide-accessible residue could correspond to Trp173 and the denaturant-resistant iodide-inaccessible one to Trp280, located in the center of the sixth transmembrane alpha-helix. Interaction of the protein with GDP (a transport inhibitor) has been studied and has revealed that it partially shields Trp173 from the interaction with I-, as well as reducing the static component of the acrylamide quenching.     

Characterization of tryptophan phosphorescence of aspartate aminotransferase from Escherichia coli.

The Trp phosphorescence spectrum, intensity and decay kinetics of apo-aspartate aminotransferase, pyridoxamine-5P-aspartate-aminotransferase and pyridoxal-5P-aspartate aminotransferase were measured over a temperature range 160-273 K. The fine structure of the phosphorescence spectra in low-temperature glasses, with 0-0 vibrational bands centered at 408, 415 and 417 nm, for both apoenzyme and pyridoxamine-5P-enzyme reveals a marked heterogeneity of the chromophore environments. Only for the pyridoxal-5P form of the enzyme is the triplet emission strongly quenched and, in this case, the spectrum displays a unique 0-0 vibrational band centered at 415 nm. Concomitant to quenching, there is Trp-sensitized delayed fluorescence of the Schiff base, an indication that quenching of the excited triplet state is due, at least in part, to a process of triplet singlet energy transfer to the ketoenamine tautomer. All three forms of the enzyme are phosphorescent for temperatures up to 273 K. However, across the glass transition temperature the pyridoxal-5P enzyme shows a decrease in lifetime-normalized phosphorescence intensity, a thermal quenching that reduces even further the number of phosphorescing residues at ambient temperature. In fluid solution, the triplet decay is nonexponential and multiple lifetimes stress the heterogeneity in dynamical structure of the chromophores' sites. For the pyridoxal-5P enzyme, where only one or at most two residues are phosphorescent at 273 K, the nonexponential nature of the decay implies the presence of different conformers of the protein not interconverting in the millisecond time scale.     

Optically detected magnetic resonance study of tyrosine residues in point-mutated bacteriophage T4 lysozyme

Two spectroscopically distinct types of tyrosine (Tyr) residues in triply point mutated bacteriophage T4 lysozyme, which contains no tryptophan (Trp), have been detected by optical detection of triplet-state magnetic resonance (ODMR) spectroscopy. Their triplet states are characterized by similar E but different D values. The Tyr site which exhibits the lower D value and has the red-shifted phosphorescence origin is quenched by energy transfer to Trp and has D and E values comparable to previously studied Tyr residues. The blue-shifted Tyr site, which is not quenched by Trp, exhibits a larger D value that has been found previously. Calculation of energy-transfer efficiencies of Tyr-Trp pairs based on the crystal structure of the native enzyme provides a possible assignment of Tyr sites to the two different spectral types.     

Conformation of amyloid fibrils of beta2-microglobulin probed by tryptophan mutagenesis

Beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis, adopts an immunoglobulin domain fold in its native state. Although beta2-m has Trp residues at positions 60 and 95, both are located near the surface of the domain. Hence, beta2-m does not have a conserved Trp common to other immunoglobulin domains, which is buried in close proximity to the disulfide bond. To study the structure of amyloid fibrils in relation to their native fold, we prepared a series of Trp mutants. Trp60 and Trp95 were both replaced with Phe, and a single Trp was introduced at various positions. Among various mutants, W39-beta2-m, in which a Trp was introduced at the position corresponding to the conserved Trp, exhibited a remarkable quenching of fluorescence in the native state, as observed for other immunoglobulin domains. An x-ray structural analysis revealed that W39-beta2-m assumes the native fold with Trp39 located in the vicinity of the disulfide bond. Comparison of the fluorescence spectra of various mutants for the native and fibrillar forms indicated that, while the Trp residues introduced in the middle of the beta2-m sequence tend to be buried in the fibrils, those located in the C-terminal region are more exposed. In addition, the fluorescence spectra of fibrils prepared at pH 2.5 and 7.0 revealed a large difference in the fluorescence intensity for W60-beta2-m, implying a major structural difference between them.     

Probing the structural diversities of long alpha-neurotoxins by fluorescence quenching studies

Trp fluorescence of Ophiophagus hannah neurotoxins (Oh-4, Oh-6A, Oh-7, and Oh-8) and Bungarus multicinctus -bungarotoxin was quenched by acrylamide and iodide. Acrylamide quenching studies indicated that the degree of exposure of Trp residues in the neurotoxins followed the order Oh-8 > Oh-7 > Oh-6A > Oh-4 > -bungarotoxin, as did the accessibility for iodide. These results reveal that the exposed degree of Trp residues and the microenvironment surrounding Trp residues in the neurotoxins differ, even though their Trp residues and positively charged residues are located at the same or homologous positions. In contrast to unfolded Oh-4, Oh-6A, Oh-7, and -bungarotoxin, unfolding of Oh-8 by reduction and S-carboxymethylation caused a notable decrease in the susceptibility of their Trp residues for iodide. These observations support the view that the side chains of Trp residues and positively charged residues in their native structure do not point toward the same spatial positions. Computer models of the neurotoxins are in good agreement with this proposition. These results elucidate why the conserved Trp residues and cationic groups do not always play the same roles in the biological activities of the neurotoxins.