Cation mediation of radical transfer between Trp48 and Tyr356 during O2 activation by protein R2 of Escherichia coli ribonucleotide reductase: relevance to R1-R2 radical transfer in nucleotide reduction?

A tryptophan 48 cation radical (W48(+)(*)) forms concomitantly with the Fe(2)(III/IV) cluster, X, during activation of oxygen for tyrosyl radical (Y122.) production in the R2 subunit of class I ribonucleotide reductase (RNR) from Escherichia coli. W48(+)(*) is also likely to be an intermediate in the long-range radical transfer between R2 and its partner subunit, R1, during nucleotide reduction by the RNR holoenzyme. The kinetics of decay of W48(+)(*) and formation of tyrosyl radicals during O(2) activation (in the absence of R1) in wild-type (wt) R2 and in variants with either Y122, Y356 (the residue thought to propagate the radical from W48(+)(*) into R1 during turnover), or both replaced by phenylalanine (F) have revealed that the presence of divalent cations at concentrations similar to the [Mg(2+)] employed in the standard RNR assay (15 mM) mediates a rapid radical-transfer equilibrium between W48 and Y356. Cation-mediated propagation of the radical from W48 to Y356 gives rise to a fast phase of Y. production that is essentially coincident with W48(+)(*) formation and creates an efficient pathway for decay of W48(+)(*). Possible mechanisms of this cation mediation and its potential relevance to intersubunit radical transfer during nucleotide reduction are considered.     

Structure and mechanism of galactose oxidase: catalytic role of tyrosine 495

 The catalytic mechanism of the copper-containing enzyme galactose oxidase involves a protein radical on Tyr272, one of the equatorial copper ligands. The first step in this mechanism has been proposed to be the abstraction of a proton from the alcohol substrate by Tyr495, the axial copper ligand that is weakly co-ordinated to copper. In this study we have generated and studied the properties of a Y495F variant to test this proposal. X-ray crystallography reveals essentially no change from wild-type other than loss of the tyrosyl hydroxyl group. Visible spectroscopy indicates a significant change in the oxidised Y495F compared to wild-type with loss of a broad 810-nm peak, supporting the suggestion that this feature is due to inter-ligand charge transfer via the copper. The presence of a peak at 420 nm indicates that the Y495F variant remains capable of radical formation, a fact supported by EPR measurements. Thus the significantly reduced catalytic efficiency (1100-fold lower k _cat / K _m) observed for this variant is not due to an inability to generate the Tyr272 radical. By studying azide-induced pH changes, it is clear that the reduced catalytic efficiency is due mainly to the inability of Y495F to accept protons. This provides definitive evidence for the key role of Tyr495 in the initial proton abstraction step of the galactose oxidase catalytic mechanism. Received: 17 December 1996 / Accepted: 12 March 1997     

The role of tryptophan 272 in the Paracoccus denitrificans cytochrome c oxidase

The mechanism of electron coupled proton transfer in cytochrome c oxidase (CcO) is still poorly understood. The P(M)-intermediate of the catalytic cycle is an oxoferryl state whose generation requires one additional electron, which cannot be provided by the two metal centres. The missing electron has been suggested to be donated to this binuclear site by a tyrosine residue. A tyrosine radical species has been detected in the P(M) and F* intermediates (formed by addition of H2O2) of the Paraccocus denitrificans CcO using electron paramagnetic resonance (EPR) spectroscopy. From the study of conserved variants its origin was determined to be Y167 which is surprising as this residue is not part of the active site. Upon inspection of the active site it becomes evident that W272 could be the actual donor of the missing electron, which can then be replenished from Y167 or from the Y280-H276 cross link in the natural cycle. To address the question, whether such a direct electron transfer pathway to the binuclear centre exists two tryptophan 272 variants in subunit I have been generated. These variants are characterised by their turnover rates as well as using EPR and optical spectroscopy. From these experiments it is concluded, that W272 is an important intermediate in the formation of the radical species appearing in P(M) and F* intermediates produced with hydrogen peroxide. The significance of this finding for the catalytic function of the enzyme is discussed.     

The iron-oxygen reconstitution reaction in protein R2-Tyr-177 mutants of mouse ribonucleotide reductase. Epr and electron nuclear double resonance studies on a new transient tryptophan radical.

The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity. We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals. In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 degrees C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in both mutants that do not have successor radicals as Trp. in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E. coli Y122F or native mouse protein R2. All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates.     

Substitution of the conserved tryptophan 31 in Escherichia coli thioredoxin by site-directed mutagenesis and structure-function analysis

All prokaryotic and eukaryotic thioredoxins contain a conserved tryptophan residue, exposed at the active site disulfide/dithiol. The role of this W31 in Escherichia coli thioredoxin (Trx) was studied by site-directed mutagenesis. Four mutant Trx with W31Y, W31F, W31H, and W31A replacements were characterized. Very low tryptophan fluorescence emission from the remaining W28 was observed in all mutant Trx; reduction resulted in large, but variable increases (up to 11-fold) of fluorescence, to levels higher than in native or denatured wild-type Trx, demonstrating a previously postulated change involving W28. All W31 mutant Trx were good substrates for E. coli thioredoxin reductase. Compared with wild type, the apparent Km values were increased less than 2-fold for the W31A, W31H, and W31F Trx and the W31Y Trx showed even slightly higher catalytic efficiency (kcat/Km value). Functions of reduced Trx with ribonucleotide reductase and in reduction of insulin disulfides were more strongly influenced by the W31 replacements, in particular at low pH for A and H residues. T7 DNA polymerase activity generated by T7 gene 5 protein and reduced Trx was lowered by large factors for W31Y, W31A, or W31H compared with W31F or the wild-type protein. The in vivo function of Trx was studied by using pUC118-trxA expression in an E. coli trxA- background. The trxA genes with W31Y and W31F substitutions restored, fully and partly, the methionine sulfoxide utilization of a trxA- metE- test strain; W31A and W31H mutations resulted in no growth. Propagation of M13 was moderately impeded by W31Y and W31F or severely by W31A and W31H replacements. Growth of a phage T3/7 hybrid was possible only with the W31Y and W31F substitutions reflecting the in vitro results for T7 DNA polymerase.     

Formation of a tyrosyl radical intermediate in Proteus mirabilis catalase by directed mutagenesis and consequences for nucleotide reactivity.

Proteus mirabilis catalase (PMC) belongs to the family of NADPH binding catalases. The function of NADPH in these enzymes is still a matter of debate. This study presents the effects of two independent phenylalanine mutations (F194 and F215), located between NADPH and heme in the PMC structure. The phenylalanines were replaced with tyrosines which we predicted could carry radicals in a NADPH-heme electron transfer. The X-ray crystal structures of the two mutants indicated that neither the binding site of NADPH nor the immediate environment of the residues was affected by the mutations. Measurements using H2O2 as a substrate confirmed that the variants were as active as the native enzyme. With equivalent amounts of peroxoacetic acid, wild-type PMC, F215Y PMC, and beef liver catalase (BLC) formed a stable compound I, while the F194Y PMC variant produced a compound I which was rapidly transformed into compound II and a tyrosyl radical. EPR studies showed that this radical, generated by the oxidation of Y194, was not related to the previously observed radical in BLC, located on Y369. In the presence of excess NADPH, compound I was reduced to a resting enzyme (k(obs) = 1.7 min(-1)) in a two-electron process. This was independent of the enzyme's origin and did not require any thus far identified tyrosyl radicals. Conversely, the presence of a tyrosyl radical in F194Y PMC greatly enhanced the oxidation of reduced beta-nicotinamide mononucleotide under a steady-state H2O2 flow with observable compound II. This process could involve a one-electron reduction of compound I via Y194.     

Evidence that amino-acid residues are responsible for substrate synergism of locust arginine kinase

A series of mutants were constructed to investigate the amino-acid residues responsible for the synergism in substrate binding of arginine kinase (AK). AK contains a pair of highly conserved amino acids (Y75 and P272) that form a hydrogen bond. In the locust (Locusta migratoria manilensis) AK, mutants in two highly conserved sites can cause pronounced loss of activity, conformational changes and distinct substrate synergism alteration. The Y75F and Y75D mutants showed strong synergism (Kd/Km=6.2-13.4), while in single mutants, P272G and P272R, and a double mutant, Y75F/P272G, the synergism was almost completely lost (Kd/Km=1.1-1.4). Another double mutant, Y75D/P272R, had characteristics similar to those of the wild-type enzyme. All these results suggest that the amino-acid residues 75 and 272 play an important role in regulating the synergism in substrate binding of AK. Fluorescence spectra showed that all mutants except Y75D/P272R displayed a red shift to different degrees. All the results provided direct evidence that there is a subtle relationship between the synergism in substrate binding and the conformational change.     

The role of a conserved tyrosine residue in high-potential iron sulfur proteins.

Conserved tyrosine-12 of Ectothiorhodospira halophila high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A). Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-11.0. Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 O delta 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-59/Val-58 peptide bond, thereby perturbing Gly-46. The delta delta GDapp of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 degrees C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 degrees C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.     

Radical sites in Mycobacterium tuberculosis KatG identified using electron paramagnetic resonance spectroscopy, the three-dimensional crystal structure, and electron transfer couplings

Catalase-peroxidase (KatG) from Mycobacterium tuberculosis, a Class I peroxidase, exhibits high catalase activity and peroxidase activity with various substrates and is responsible for activation of the commonly used antitubercular drug, isoniazid (INH). KatG readily forms amino acid-based radicals during turnover with alkyl peroxides, and this work focuses on extending the identification and characterization of radicals forming on the millisecond to second time scale. Rapid freeze-quench electron paramagnetic resonance spectroscopy (RFQ-EPR) reveals a change in the structure of the initially formed radical in the presence of INH. Heme pocket binding of the drug and knowledge that KatG[Y229F] lacks this signal provides evidence for radical formation on residue Tyr(229). High field RFQ-EPR spectroscopy confirmed a tryptophanyl radical signal, and new analyses of X-band RFQ-EPR spectra also established its presence. High field EPR spectroscopy also confirmed that the majority radical species is a tyrosyl radical. Site-directed mutagenesis, along with simulations of EPR spectra based on x-ray structural data for particular tyrosine and tryptophan residues, enabled assignments based on predicted hyperfine coupling parameters. KatG mutants W107F, Y229F, and the double mutant W107F/Y229F showed alteration in type and yield of radical species. Results are consistent with formation of a tyrosyl radical reasonably assigned to residue Tyr(229) within the first few milliseconds of turnover. This is followed by a mixture of tyrosyl and tryptophanyl radical species and finally to only a tyrosyl radical on residue Tyr(353), which lies more distant from the heme. The radical processing of enzyme lacking the Trp(107)-Tyr(229)-Met(255) adduct (found as a unique structural feature of catalase-peroxidases) is suggested to be a reasonable assignment of the phenomena.     

The role of the conserved tryptophan272 of the Paracoccus denitrificans cytochrome c oxidase in proton pumping

The catalytic mechanism of heme-copper oxidases - electron transfer coupled to proton pumping - is not yet fully understood. Single turnover experiments in which fully reduced cytochrome aa(3) from Paracoccus denitrificans reacts with O(2) using the microsecond freeze-hyperquenching sampling technique enabled trapping of transient catalytic intermediates and analysis by low temperature UV-Visible, X-band and Q-band EPR spectroscopy. Our recent findings (Wiertz et al. (2007) J. Biol. Chem. 282, 31580-31591), which show that the strictly conserved W272 is a redox active residue are reviewed here. The W272 forms a tryptophan neutral radical in the transition F-->F(W)-->O(H) in which the novel intermediate F(W) harbors the tryptophan radical. The potential role of W272 in proton pumping is highlighted.     

Cytochrome b5 reductase: role of the si-face residues, proline 92 and tyrosine 93, in structure and catalysis

The conserved sequence motif "RxY(T)(S)xx(S)(N)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(T)(S)xx(S)(N)" motif, that stacks against the si-face of the flavin isoalloxazine ring, and P92, the second residue in the motif that is also in close proximity to the FAD moiety, a series of rat cb(5)r variants were produced with substitutions at either P92 or Y93, respectively. The proline mutants P92A, G, and S together with the tyrosine mutants Y93A, D, F, H, S, and W were recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein was found to bind FAD in a 1:1 cofactor:protein stoichiometry while UV CD spectra suggested similar secondary structure organization among all nine variants. The tyrosine variants Y93A, D, F, H, and S exhibited varying degrees of blue-shift in the flavin visible absorption maxima while visible CD spectra of the Y93A, D, H, S, and W mutants exhibited similar blue-shifted maxima together with changes in absorption intensity. Intrinsic flavin fluorescence was quenched in the wild type, P92S and A, and Y93H and W mutants while Y93A, D, F, and S mutants exhibited increased fluorescence when compared to free FAD. The tyrosine variants Y93A, D, F, and S also exhibited greater thermolability of FAD binding. The specificity constant (k(cat)/K(m)(NADH)) for NADH:FR activity decreased in the order wild type > P92S > P92A > P92G > Y93F > Y93S > Y93A > Y93D > Y93H > Y93W with the Y93W variant retaining only 0.5% of wild-type efficiency. Both K(s)(H4NAD) and K(s)(NAD+) values suggested that Y93A, F, and W mutants had compromised NADH and NAD(+) binding. Thermodynamic measurements of the midpoint potential (E degrees ', n = 2) of the FAD/FADH(2) redox couple revealed that the potentials of the Y93A and S variants were approximately 30 mV more positive than that of wild-type cb(5)r (E degrees ' = -268 mV) while that of Y93H was approximately 30 mV more negative. These results indicate that neither P92 nor Y93 are critical for flavin incorporation in cb(5)r and that an aromatic side chain is not essential at position 93, but they demonstrate that Y93 forms contacts with the FAD that effectively modulate the spectroscopic, catalytic, and thermodynamic properties of the bound cofactor.     

The conserved Trp-Cys hydrogen bond dampens the "push effect" of the heme cysteinate proximal ligand during the first catalytic cycle of nitric oxide synthase

Residues surrounding and interacting with the heme proximal ligand are important for efficient catalysis by heme proteins. The nitric oxide synthases (NOSs) are thiolate-coordinated enzymes that catalyze the hydroxylation of l-Arg in the first of the two catalytic cycles needed to synthesize nitric oxide. In NOSs, the indole NH group of a conserved tryptophan [W56 of the bacterial NOS-like protein from Staphylococcus aureus (saNOS)] forms a hydrogen bond with the heme proximal cysteinate ligand. The purpose of this study was to determine the impact of increasing (W56F and W56Y variants) or decreasing (W56H variant) the electron density of the proximal cysteinate ligand on molecular oxygen (O(2)) activation using saNOS as a model. We show that the removal of the indole NH···S(-) bond for W56F and W56Y caused an increase in the electron density of the cysteinate. This was probed by the decrease of the midpoint reduction potential (E(1/2)) along with weakened σ-bonding and strengthened π-backbonding with distal ligands (CO and O(2)). On the other hand, the W56H variant showed stronger Fe-OO and Fe-CO bonds (strengthened σ-bonding) along with an elevated E(1/2), which is consistent with the formation of a strong NH···S(-) hydrogen bond from H56. We also show here that changing the electron density of the proximal thiolate controls its "push effect"; whereas the rates of both O(2) activation and autoxidation of the Fe(II)O(2) complex increase with the stronger push effect created by removing the indole NH···S(-) hydrogen bond (W56F and W56Y variants), the W56H variant showed an increased stability of the complex against autoxidation and a slower rate of O(2) activation. These results are discussed with regard to the roles played by the conserved tryptophan-cysteinate interaction in the first catalytic cycle of NOS.     

Use of a chemical trigger for electron transfer to characterize a precursor to cluster X in assembly of the iron-radical cofactor of Escherichia coli ribonucleotide reductase

A key step in generation of the catalytically essential tyrosyl radical (Y122(*)) in protein R2 of Escherichia coli ribonucleotide reductase is electron transfer (ET) from the near-surface residue, tryptophan 48 (W48), to a (Fe(2)O(2))(4+) complex formed by addition of O(2) to the carboxylate-bridged diiron(II) cluster. Because this step is rapid, the (Fe(2)O(2))(4+) complex does not accumulate and, therefore, has not been characterized. The product of the ET step is a "diradical" intermediate state containing the well-characterized Fe(IV)Fe(III) cluster, X, and a W48 cation radical (W48(+)(*)). The latter may be reduced from solution to complete the two-step transfer of an electron to the buried diiron site. In this study, a (Fe(2)O(2))(4+) state that is probably the precursor to the X-W48(+)(*) diradical state in the reaction of the wild-type protein (R2-wt) has been characterized by exploitation of the observation that in R2 variants with W48 replaced with alanine (A), the otherwise disabled ET step can be mediated by indole compounds. Mixing of the Fe(II) complex of R2-W48A/Y122F with O(2) results in accumulation of an intermediate state that rapidly converts to X upon mixing with 3-methylindole (3-MI). The state comprises at least two species, of which each exhibits an apparent M?ssbauer quadrupole doublet with parameters characteristic of high-spin Fe(III) ions. The isomer shifts of these complexes and absence of magnetic hyperfine coupling in their M?ssbauer spectra suggest that both are antiferromagnetically coupled diiron(III) clusters. The fact that both rapidly convert to X upon treatment with a molecule (3-MI) shown in the preceding paper to mediate ET in W48A R2 variants indicates that they are more oxidized than X by one electron, which suggests that they have a bound peroxide equivalent. Their failure to exhibit either the long-wavelength absorption (at 650-750 nm) or M?ssbauer doublet with high isomer shift (>0.6 mm/s) that are characteristic of the putatively mu-1,2-peroxo-bridged diiron(III) intermediates that have been detected in the reactions of methane monooxygenase (P or H(peroxo)) and variants of R2 with the D84E ligand substitution suggests that they have geometries and electronic structures different from those of the previously characterized complexes. Supporting this deduction, the peroxodiiron(III) complex that accumulates in R2-W48A/D84E is much less reactive toward 3-MI-mediated reduction than the (Fe(2)O(2))(4+) state in R2-W48A/Y122F. It is postulated that the new (Fe(2)O(2))(4+) state is either an early adduct in an orthogonal pathway for oxygen activation or, more likely, the successor to a (mu-1,2-peroxo)diiron(III) complex that is extremely fleeting in R2 proteins with the wild-type ligand set but longer lived in D84E-containing variants.     

Variants of DNA polymerase Beta extend mispaired DNA due to increased affinity for nucleotide substrate

DNA polymerase beta offers an attractive system to study the biochemical mechanism of polymerase-dependent mutagenesis. Variants of DNA polymerase beta, Y265F and Y265W, were analyzed for misincorporation efficiency and mispair extension ability, relative to wild-type DNA polymerase beta. Our data show that the fidelity of the mutant polymerases is similar to wild-type enzyme on a one-nucleotide gapped DNA substrate. In contrast, with a six-nucleotide gapped DNA, the mutant proteins are slightly more accurate than the wild-type enzyme. The mutagenic potential of Y265F and Y265W is more pronounced when encountering a mispaired DNA substrate. Here, both variants can extend a G:G mispair quite efficiently, and Y265F can also extend a T:G mispair. The kinetic basis of the increased mispair extension efficiency is due to an improved ability to bind to the incoming nucleotide. Y265W extends the G:G mispair even with an incorrect nucleotide substrate. Overall, our results demonstrate that the Y265 hinge residue is important for stabilizing the architecture of the nucleotide binding pocket of DNA polymerase beta, and that alterations of this residue can have significant impacts upon the fidelity of DNA synthesis.     

Sugar binding to recombinant wild-type and mutant glucokinase monitored by kinetic measurement and tryptophan fluorescence

Tryptophan fluorescence was used to study GK (glucokinase), an enzyme that plays a prominent role in glucose homoeostasis which, when inactivated or activated by mutations, causes diabetes mellitus or hypoglycaemia in humans. GK has three tryptophan residues, and binding of D-glucose increases their fluorescence. To assess the contribution of individual tryptophan residues to this effect, we generated GST-GK [GK conjugated to GST (glutathione transferase)] and also pure GK with one, two or three of the tryptophan residues of GK replaced with other amino acids (i.e. W99C, W99R, W167A, W167F, W257F, W99R/W167F, W99R/W257F, W167F/W257F and W99R/W167F/W257F). Enzyme kinetics, binding constants for glucose and several other sugars and fluorescence quantum yields (varphi) were determined and compared with those of wild-type GK retaining its three tryptophan residues. Replacement of all three tryptophan residues resulted in an enzyme that retained all characteristic features of GK, thereby demonstrating the unique usefulness of tryptophan fluorescence as an indicator of GK conformation. Curves of glucose binding to wild-type and mutant GK or GST-GK were hyperbolic, whereas catalysis of wild-type and most mutants exhibited co-operativity with D-glucose. Binding studies showed the following order of affinities for the enzyme variants: N-acetyl-D-glucosamine>D-glucose>D-mannose>D-mannoheptulose>2-deoxy-D-glucose>L-glucose. GK activators increased sugar binding of most enzymes, but not of the mutants Y214A/V452A and C252Y. Contributions to the fluorescence increase from Trp(99) and Trp(167) were large compared with that from Trp(257) and are probably based on distinct mechanisms. The average quantum efficiency of tryptophan fluorescence in the basal and glucose-bound state was modified by activating (Y214A/V452A) or inactivating (C213R and C252Y) mutations and was interpreted as a manifestation of distinct conformational states.     

Membrane interaction and activity of the glycolipid transfer protein

In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.     

Membrane interaction and activity of the glycolipid transfer protein

In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.     

Characterization of two mutant lactose repressor proteins containing single tryptophans

Two mutant lactose repressors, each containing a single tryptophan, were generated by site-specific mutagenesis. Tyrosine was substituted for tryptophan to be analogous to amber suppression mutants reported previously (Sommer, H., Lu, P., and Miller, J. H. (1976) J. Biol. Chem. 251, 3774-3779). Unlike the amber suppression mutants, plasmids containing the mutant sequences produce large quantities of stable, easily isolable protein. The binding properties of the site-specific mutant repressors (W201Y, W220Y) differ from those reported for the corresponding suppression mutants (A201, A220). Whereas minimal effects on operator dissociation rate from lambda plac DNA were noted for the suppression mutants, purified W201Y and W220Y proteins exhibit 10- and 5-fold reduced affinity for a 40-base pair operator, respectively, compared with wild-type. Inducer binding of the A201 and W201Y mutants was similar to that for wild-type repressor, but the inducer affinity of W220Y was approximately 2-fold lower than A220 (approximately 30-fold lower than wild-type). Fluorescence spectra and iodide quenching of the mutant proteins were similar to the suppression mutants, but the absorption coefficient differed significantly from the values reported previously. Acrylamide and iodide quenching results indicate that Trp201 is relatively buried whereas Trp220 is exposed to solvent; inducer binding reduces quenching of Trp220 significantly. CD spectra indicate that the mutant proteins have secondary structural features similar to those of wild-type. Inducer UV difference spectra showed that the major features reported for the wild-type isopropyl beta-D-thiogalactopyranoside difference spectrum were attributable to both tryptophans. In the presence of melibiose, a new minimum appeared in the difference spectra of wild-type and W201Y which was not evident when these proteins bound isopropyl beta-D-thiogalactopyranoside. It is possible that this new feature results from Trp220 involvement in a direct contact with the second sugar in disaccharide inducer molecules such as melibiose and 1,6-allolactose.     

Early events during folding of wild-type staphylococcal nuclease and a single-tryptophan variant studied by ultrarapid mixing

A continuous-flow mixing device with a dead time of 100 micros coupled with intrinsic tryptophan and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was used to monitor structure formation during early stages of the folding of staphylococcal nuclease (SNase). A variant with a unique tryptophan fluorophore in the N-terminal beta-barrel domain (Trp76 SNase) was obtained by replacing the single Trp140 in wild-type SNase with His in combination with Trp substitution of Phe76. A common background of P47G, P117G and H124L mutations was chosen in order to stabilize the protein and prevent accumulation of cis proline isomers under native conditions. In contrast to WT(*) SNase, which shows no changes in tryptophan fluorescence prior to the rate-limiting folding step ( approximately 100 ms), the F76W/W140H variant shows additional changes (enhancement) during an early folding phase with a time constant of 75 micros. Both proteins exhibit a major increase in ANS fluorescence and identical rates for this early folding event. These findings are consistent with the rapid accumulation of an ensemble of states containing a loosely packed hydrophobic core involving primarily the beta-barrel domain while the specific interactions in the alpha-helical domain involving Trp140 are formed only during the final stages of folding. The fact that both variants exhibit the same number of kinetic phases with very similar rates confirms that the folding mechanism is not perturbed by the F76W/W140H mutations. However, the Trp at position 76 reports on the rapid formation of a hydrophobic cluster in the N-terminal beta-sheet region while the wild-type Trp140 is silent during this early stage of folding. Quantitative modeling of the (un)folding kinetics and thermodynamics of these two proteins versus urea concentration revealed that the F76W/W140H mutation selectively destabilizes the native state relative to WT(*) SNase while the stability of transient intermediates remains unchanged, leading to accumulation of intermediates under equilibrium conditions at moderate denaturant concentrations.     

Mechanism of thermostability in thermolysin – analysis of subsite S2 mutant enzymes of thermolysin

An aromatic amino acid at position 115 (tryptophan residue; subsite S2) in thermolysin is known to be essential for proteolytic activity of thermolysin. Mutant enzymes substituted by phenylalanine (W115F) and tyrosine (W115Y) at position 115 were expressed at similar levels as the wild type (WT) enzyme in Bacillus subtilis . The thermostability of the W115Y mutant enzyme was equal to that of the WT. However, that of the W115F mutant enzyme was significantly lower than the WT. Enzymatic kcat/Km values of W115F increased to about twice those of the WT, but W115F also seemed to promote increased autodegradation compared with the WT and W115Y enzymes.