Tryptophan properties in fluorescence and functional stability of plasminogen activator inhibitor 1

Plasminogen activator inhibitor 1 harbors four tryptophan residues at positions 86, 139, 175, and 262. To investigate the contribution of each tryptophan residue to the total fluorescence and to reveal the mutual interactions of the tryptophan residues and interactions with the other amino acids, 15 mutants in which tryptophan residues have been replaced by phenylalanines were constructed, purified, and characterized. Conformational distribution analysis revealed that the tryptophan mutants have a similar conformational distribution pattern as wild-type plasminogen activator inhibitor 1. Mutants in which tryptophan residue 175 was replaced by a phenylalanine displayed an increased functional half-life of the active conformation, whereas the functional half-life of mutants in which tryptophan residue 262 was replaced by a phenylalanine was substantially decreased. Comparative analysis of the fluorescence lifetimes, the extinction coefficients, and the quantum yields of the individual tryptophan residues demonstrates that tryptophan residue 262 gives the highest contribution to the total fluorescence. The other tryptophan residues have a very low quantum yield. In the wild-type protein, the fluorescence of all tryptophan residues is partially quenched as compared to the mutants that contain single tryptophan residues, due to conformational effects. The fluorescence of tryptophan residue 262 is very likely also partially quenched by energy transfer to tryptophan residue 175.     

Identification of the Tryptophan Residue Located at the Saccharide Binding Site of Castor Bean Hemagglutinin

The tryptophan residue present at the saccharide-binding site of castor bean hemagglutinin (CBH) was identified. A peptide containing a modified tryptophan residue was isolated from the tryptic digest of S-car boxy methylated B-chain obtained from an inactive derivative of CBH (2-Oxa-CBH), in which two tryptophan residues/mol were oxidized with Af-bromosuccinimide, by gel filtration on a Sephadex G-50 followed by high performance liquid chromatography. Analytical data for the isolated peptide indicated that the tryptophan residue at position 131 on the B-chain was modified in 2-Oxa-CBH.

From these and earlier results, it is suggested that the tryptophan residue at 131 on each B-chain is closely associated with the saccharide-binding activity of CBH. The specific role of tryptophan residue at 131 in the saccharide-binding site of CBH is also discussed.     

Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana

The location of tryptophan residues in the actin macromolecule was studied on the basis of the known 3D structure. For every tryptophan residue the polarity and packing density of their microenvironments were evaluated. To estimate the accessibility of the tryptophan residues to the solvent molecules it was proposed to analyze the radial dependence of the packing density of atoms in the macromolecule about the geometric center of the indole rings of the tryptophan residues. The proposed analysis revealed that the microenvironment of tryptophan residues Trp-340 and Trp-356 has a very high density. So these residues can be regarded as internal and inaccessible to solvent molecules. Their microenvironment is mainly formed by non-polar groups of protein. Though the packing density of the Trp-86 microenvironment is lower, this tryptophan residue is apparently also inaccessible to solvent molecules, as it is located in the inner region of macromolecule. Tryptophan residue Trp-79 is external and accessible to the solvent. All residues that can affect tryptophan fluorescence were revealed. It was found that in the close vicinity of tryptophan residues Trp-79 and Trp-86 there are a number of sulfur atoms of cysteine and methionine residues that are known to be effective quenchers of tryptophan fluorescence. The most essential is the location of SG atom of Cys-10 near the NE1 atom of the indole ring of tryptophan residue Trp-86. On the basis of microenvironment analysis of these tryptophan residues and the evaluation of energy transfer between them it was concluded that the contribution of tryptophan residues Trp-79 and Trp-86 must be low. Intrinsic fluorescence of actin must be mainly determined by two other tryptophan residues--Trp-340 and Trp-356. It is possible that the unstrained conformation of tryptophan residue Trp-340 and the existence of aromatic rings of tyrosine and phenylalanine and proline residues in the microenvironments of tryptophan residues Trp-340 and Trp-356 are also essential to their blue fluorescence spectrum.     

Significance of the Tryptophan Residue at the Low Affinity Saccharide-binding Site of Ricin E

Chemical modification of tryptophan residues in ricin E was investigated with regard to saccharide-binding. Two out of ten tryptophan residues in ricin E were modified with N- bromosuccinimide at pH 4.5 in the absence of specific saccharide accompanied by a marked decrease in the cytoagglutinating activity. Such a loss of the cytoagglutinating activity was found to be principally due to the oxidation of one tryptophan residue per B-chain. In the presence of lactose, one tryptophan residue/mol was protected from the modification with retention of a fairly high cytoagglutinating activity. However, G a IN Ac did not show such a protective effect. The binding of lactose to ricin E altered the environment of the tryptophan residue at the low affinity binding site of ricin E, leading to a blue shift of the fluorescence spectrum and an UV-difference spectrum with a maximum at 290 nm and a trough at 300 nm. The ability to generate such spectroscopic changes induced by lactose was retained in the derivative in which one tryptophan residue/mol was oxidized in the presence of lactose, but not in the derivative in which two tryptophan residues/mol were oxidized in the absence of lactose. Based on these results, it is suggested that one of the two surface-localized tryptophan residues is responsible for saccharide binding at the low affinity binding site of ricin E, which can bind lactose but lacks the ability to bind GalNAc.     

Role of tryptophan hydroxylase phe313 in determining substrate specificity

The active site residue phenylalanine 313 is conserved in the sequences of all known tryptophan hydroxylases. The tryptophan hydroxylase F313W mutant protein no longer shows a preference for tryptophan over phenylalanine as a substrate, consistent with a role of this residue in substrate specificity. A tryptophan residue occupies the homologous position in tyrosine hydroxylase. The tyrosine hydroxylase W372F mutant enzyme does not show an increased preference for tryptophan over tyrosine or phenylalanine, so that this residue cannot be considered the dominant factor in substrate specificity in this family of enzymes.     

Correlation between internal motion and emission kinetics of tryptophan residues in proteins

Time-resolved fluorescence anisotropy measurements of tryptophan residues were carried out for 44 proteins. Internal rotational motion with a sub-nanosecond correlation time (0.9 +/- 0.6 ns at 10 degrees C) was seen in a large number of proteins, though its amplitude varied from protein to protein. It was found that tryptophan residues which were almost fixed within a protein had either a long (greater than 4 ns) or short (less than 2 ns) fluorescence lifetime, whereas a residue undergoing a large internal motion had an intermediate lifetime (1.5-3 ns). It is suggested that the emission kinetics of a tryptophan residue is coupled with its internal motion. In particular, an immobile tryptophan residue emitting at long wavelength was characterized by a long lifetime (greater than 4 ns). It appears that a tryptophan residue fixed in a polar region has little chance of being quenched by neighboring groups.     

Nanosecond fluorometry of the single tryptophan in cytochrome P-450e (P450IIB2)

Properties of the single tryptophan residue in rat liver microsomal phenobarbital-inducible cytochrome P-450e (P450IIB2) were studied by the nanosecond time-resolved fluorometry. The tryptophan fluorescence decay time was found to be 3.6 ns and it was not affected by the addition of substrate (perhydrophenanthrene). This result strongly indicates that the tryptophan residue is not a part of the substrate-binding site.     

Nanosecond motions of the single tryptophan residues in apolipoproteins C-I and C-II: a study by oxygen quenching and fluorescence depolarization

Rotational freedom of the single tryptophan residue in human plasma apolipoproteins C-I (apo C-I) and C-II (apo C-II) was investigated by oxygen quenching and lifetime-resolved anisotropies. The tryptophan in both apo C-I and C-II was highly accessible to oxygen quenching. The tryptophan residue in both apo C-I and C-II and their sodium dodecyl sulfate (SDS) or dimyristoylphosphatidylcholine (DMPC) complexes displayed significant motional freedom on the nanosecond time scale. Lifetime-resolved anisotropies of tryptophan residues under conditions of oxygen quenching revealed an increase in the amplitude of the segmental motions at 40 degrees C as compared to that at 5 degrees C. It was concluded from these studies that both the apoprotein C-I and C-II are highly flexible molecules, and that the nanosecond motions of the tryptophan residue are sensitive to the fluidity of its environment in both SDS and DMPC complexes.     

Geranyl modification on the tryptophan residue of ComXRO-E-2 pheromone by a cell-free system

ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in the gram-positive bacterium Bacillus. Since posttranslational prenylation on the tryptophan residue has not been reported except in ComX pheromone, the universality of this modification has not yet been elucidated. In this paper, we established a cell-free system, whereby the tryptophan residue in peptides is modified with a geranyl group by modifying enzyme ComQ. In addition, we investigated enzymatic reaction conditions using an in vitro enzyme reaction system. This is the first report of in vitro geranylation on the tryptophan residue. This system is potentially a useful tool for elucidating the universality of prenylation on the tryptophan residue.     

Synthesis and biological activity of 2-phenylethyl ester analogues of C-terminal heptapeptide of cholecystokinin modified in Trp 30 region.

We have tried to evaluate the significance of the tryptophan side chain residue and of the surrounding peptide bonds in the antagonist activity of cholecystokinin analogues lacking the C-terminal amide function and having a D-tryptophan. In order to perform this study, analogues of the C-terminal heptapeptide of cholecystokinin were synthesized by replacing the C-terminal phenylalanine residue with 2-phenylethyl alcohol and by either replacing the tryptophan residue with an alanine, a norleucine and a phenylalanine residue, or introducing a "reduced peptide bond" in the tryptophan 30 region. Most of these compounds were able to reproduce only part of the response of cholecystokinin in stimulating amylase release from rat pancreatic acini, as was already observed for 2-phenylethyl ester analogues of CCK. These results point out the key role of tryptophan 30 in the biological response of cholecystokinin.     

The modification of the lone tryptophan residue in human serum albumin by 2-hydroxy-5-nitrobenzyl bromide. Characterization of the modified protein and the binding of L-tryptophan and benzodiazepines to the tryptophan-modified albumin.

The possible function of the lone tryptophan residue of human serum albumin in the stereospecific binding site for indole and benzodiazepine compounds was investigated by chemical modification. This residue can be selectively modified with 2-hydroxy-5-nitrobenzyl bromide. The modification alters the conformation of the albumin only slightly, as revealed by circular dichroism, fluorescence, and ultraviolet absorption measurements. A decrease in the association constants of L-tryptophan and diazepam of about 30 - 50% and a decrease in the extrinsic Cotton effects of four benzodiazepine derivatives of about 10 - 15% were found as specific effects of the tryptophan modification. The tryptophan modification itself did not change the number of binding sites of diazepam and L-tryptophan. It is suggested that the lone tryptophan residue of human serum albumin is not directly involved in the specific binding site for indole and benzodiazepine compounds. However, the modification alters the properties of the binding site either by an incomplete refolding of the albumin after urea treatment, or a more selective allosteric effect of the modified tryptophan residue.     

Fluorescence of tryptophan residues in firefly luciferases and enzyme--substrate complexes

Quenching of tryptophan fluorescence of Luciola mingrelica (single tryptophan residue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417 and Trp-426) luciferases with different quenchers (I-, Cs+, acrylamide) was studied. The conserved Trp-417(419) residue was shown to be not accessible to charged particles, and positively and negatively charged amino acid residues are located in close vicinity to it. We found previously unreported effective energy transfer from this tryptophan to luciferin during the quenching of the tryptophan fluorescence. The distance between the luciferin molecule and Trp-417(419) was calculated: 11-15 and 12-17 A for P. pyralis and L. mingrelica luciferases, respectively. The role of the conserved Trp residue in the catalysis is discussed. ATP and AMP are also quenchers of the tryptophan fluorescence of the luciferases. In this case, an allosteric mechanism of the interaction of Trp-417(419) with an excess of ATP (AMP) is proposed.     

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.     

Role of the tryptophan residue in the vicinity of the catalytic center of exonuclease III family AP endonucleases: AP site recognition mechanism

The mechanisms by which AP endonucleases recognize AP sites have not yet been determined. Based on our previous study with Escherichia coli exonuclease III (ExoIII), the ExoIII family AP endonucleases probably recognize the DNA-pocket formed at an AP site. The indole ring of a conserved tryptophan residue in the vicinity of the catalytic site presumably intercalates into this pocket. To test this hypothesis, we constructed a series of mutants of ExoIII and human APE1. Trp-212 of ExoIII and Trp-280 of APE1 were critical to the AP endonuclease activity and binding to DNA containing an AP site. To confirm the ability of the tryptophan residue to intercalate with the AP site, we examined the interaction between an oligopeptide containing a tryptophan residue and an oligonucleotide containing AP sites, using spectrofluorimetry and surface plasmon resonance (SPR) technology. The tryptophan residue of the oligopeptide specifically intercalated into an AP site of DNA. The tryptophan residue in the vicinity of the catalytic site of the ExoIII family AP endonucleases plays a key role in the recognition of AP sites.     

Identification of the tryptophan residue located at the low-affinity saccharide binding site of ricin D

The saccharide binding ability of the low affinity (LA-) binding site of ricin D was abrogated by N-bromosuccinimide (NBS)-oxidation, while in the presence of lactose the number of tryptophan residues eventually oxidized decreased by 1 mol/mol and the saccharide binding ability was retained (Hatakeyama et al., (1986) J. Biochem. 99, 1049-1056). Based on these findings, the tryptophan residue located at the LA-binding site of ricin D was identified. Two derivatives of ricin D which were modified with NBS in the presence and absence of lactose were separated into their constituent polypeptide chains (A- and B-chains), respectively. The modified tryptophan residue or residues was/were found to be contained in the B-chain, but not in the A-chain. From lysylendopeptidase and chymotryptic digests, peptides containing oxidized tryptophan residues were isolated by gel filtration on Bio-Gel P-30 and HPLC. Analysis of the peptides containing oxidized tryptophan revealed that three tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative of ricin D, in which the saccharide binding ability of the LA-binding site was abrogated by NBS-oxidation. On the other hand, the modified residues were determined to be tryptophans at positions 93 and 160 in the active derivative of ricin D which was modified in the presence of lactose, indicating that upon binding with lactose, the tryptophan residue at position 37 of the B-chain was protected from NBS-oxidation. From these results, it is suggested that tryptophan at position 37 on the B-chain is the essential residue for saccharide binding at the LA-binding site of ricin D.     

Identification of the Tryptophan Residue Located at the Substrate-binding Site of Rye Seed Chitinase-c

Chemical modifications of rye seed chitinase-c (RSC-c) with various reagents suggested the involvements of tryptophan and glutamic/aspartic acid residues in the activity. Of these, the modification of tryptophan residues with N-bromosuccinimide (NBS) was investigated in detail.

In the NBS-oxidation at pH 4.0, two of the six tryptophan residues in RSC-c were rapidly oxidized and the chitinase activity was almost completely lost. On the other hand, in the NBS-oxidation at pH 5.9, only one tryptophan residue was oxidized and the activity was greatly reduced. Analyses of the oxidized tryptophan-containing peptides from the tryptic and chymotryptic digests of the modified RSC-c showed that two tryptophan residues oxidized at pH 4.0 are Trp72 and Trp82, and that oxidized at pH 5.9 is Trp72.

The NBS-oxidation of Trp72 at pH 5.9 was protected by a tetramer of N-acetylglucosamine (NAG₄), a very slowly reactive substrate for RSC-c, and the activity was almost fully retained. In the presence of NAG4, RSC-c exhibited an UV -difference spectrum with maxima at 284 nm and 293 nm, attributed to the red shift of the tryptophan residue, as well as a small trough around 300 nm probably due to an alteration of the environment of the tryptophan residue. From these results, it was suggested that Trp72 is exposed on the surface of the RSC-c molecule and involved in the binding to substrate.     

Evidence for the involvement of tryptophan 38 in the active site of glutathione S-transferase P.

Glutathione S-transferase P (GST-P) exists as a homodimeric form and has two tryptophan residues, Trp28 and Trp38, in each subunit. In order to elucidate the role of the two tryptophan residues in catalytic function, we examined intrinsic fluorescence of tryptophan residues and effect of chemical modification by N-bromosuccinimide (NBS). The quenching of intrinsic fluorescence was observed by the addition of S-hexylglutathione, a substrate analogue, and the enzymatic activity was totally lost when single tryptophan residue was oxidized by NBS. To identify which tryptophan residue is involved in the catalytic function, each tryptophan was changed to histidine by site-directed mutagenesis. Trp28His GST-P mutant enzyme showed a comparable enzymatic activity with that of the wild type one. Trp38His mutant neither was bound to S-hexylglutathione-linked Sepharose nor exhibited any GST activity. These findings indicate that Trp38 is important for the catalytic function and substrate binding of GST-P.     

Participation of the lone tryptophan residue of rat alpha-foetoprotein in its drug-binding sites. Comparison with rat serum albumin.

The participation in drug binding of the lone tryptophan residue of rat alpha-foetoprotein (alpha-FP) and serum albumin, the two main transport proteins of foetal serum, has been studied by two different techniques. Firstly, the effect on phenylbutazone and warfarin binding of the chemical derivatization of the lone tryptophan residue of both proteins by 2-nitrophenylsulphonyl chloride (NPS) was studied. Secondly, the effect of phenylbutazone binding on the intrinsic fluorescence of the tryptophan residue of rat alpha-FP and albumin was investigated. The specific modification of the proteins by NPS did not affect the binding of warfarin by rat alpha-FP and albumin, but greatly decreased the affinity of the high-affinity sites of rat alpha-FP for phenylbutazone, though the numbers of these sites were not significantly changed. However, for albumin a similar decrease in the affinity constant appeared to be due to the reaction conditions. The spectrofluorimetric studies showed that the lone tryptophan residue of alpha-FP and albumin was quenched by phenylbutazone binding, and the quenching paralleled the fractional saturation of the high-affinity site only in the case of albumin. The effect of phenylbutazone binding on the intrinsic fluorescence of rat alpha-FP indicated that the lone tryptophan residue of this foetal protein is not in the same molecular environment as that of albumin, not participating directly in the high-affinity site for phenylbutazone, and the effect may be via some induced conformational change in rat alpha-FP. These results also confirm our previous suggestion that the high-affinity sites for phenylbutazone and warfarin are different on the rat alpha-FP molecule. The results seem to indicate that this is also the case for albumin, but confirmation is necessary.     

The pH dependence of binding of alpha,alpha'-dibromo-p-xylenesulfonic acid to lysozyme.

The chemical modification of lysozyme (I) has been accomplished with alpha, alpha'-dibromo-p-xylenesulfonic acid (DBX) at five different pH values. I was alkylated by DBX at room temperature (28 degrees C) with decrease in enzyme activity. The rate of inactivation depended upon the pH at which alkylation was carried out. The highest rate was seen at alkaline pH values; the lowest at more acidic pH values. Amino acid analyses showed that-two lysines and two tryptophan residues had been modified at pH 9; two lysines, one tryptophan and one methionine had reacted at pH 8. A histidine residue was bound at pH 6.5 together with a tryptophan residue. At the lower pH values (2.7, 4.5, 6.5), alkylation occurred with a single tryptophan residue each. Fluorescence and CD data both ruled out the participation of tryptophans 62 or 108. Labeling experiments showed that two residues of DBX-35S were bound per molecule of I at both pH9 and pH8; one residue of DBX was bound per molecule of I at the other pH values. Sedimentation coefficients were characteristic of native lysozyme. The stoichiometry of binding and residue modification indicated that intra-molecular cross links were established. The pH dependence of the cross-linking provides means to measure several allowed intra-molecular distances. The results presented here are consistent with the existence of side chain motion in lysozyme.     

The role of an invariant tryptophan residue in alpha-bungarotoxin and cobrotoxin. Investigation of active derivatives with the invariant tryptophan replaced by kynurenine

Ozone oxidation converted the single, invariant, tryptophan residue to N2-formylkynurenine in alpha-bungarotoxin and cobrotoxin. Upon this modification, the lethal toxicity was significantly reduced in cobrotoxin but mostly retained in alpha-bungarotoxin. Each neurotoxin containing kynurenine instead of tryptophan retained the same antigenicity as the native toxin. Fluorescence and CD spectroscopy revealed that, although the environment and state of the kynurenine residue were similar, [Kyn29]cobrotoxin was much more sensitive to pH change than alpha-[Kyn28]bungarotoxin. In terms of lethal toxicity and conformational stability, the invariant tryptophan residue appears to play a more important role in cobrotoxin, imparting a higher lethal toxicity than that in alpha-bungarotoxin, which has a disulfide bond at Cys29-Cys33.