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1.
We have determined the picosecond fluorescence of the four aromatic amino acid residues (W28, W31, Y49, and Y70) in wild-type Escherichia coli thioredoxin (wt Trx) and a mutant Trx with W31 replaced by phenylalanine, Trx-W28-W31F. The internal motions of the four aromatic side chains were also analyzed. We examined the possibility of using internal energy transfer from tyrosine to tryptophan as a measure of long-range distances. The major features of the lifetime distribution of tryptophan fluorescence were unchanged in the W31F mutation, indicating that the environment of W28 is similar in both wt Trx and Trx-W28-W31F. However, the mutation of W31F changed the mobility of W28, situated close to the active-site disulfide/dithiol, but not the mobility of two tyrosines, Y49 and Y70, situated on the other side of the molecule. The mobility of the two tyrosine residues increased upon reduction of the active-site disulfide, indicating a looser structure with reduction. This increased motion could also be seen from molecular dynamics simulations. The change in energy transfer rates, as judged by tyrosine fluorescence lifetimes, was in agreement with energy transfer rates calculated from the molecular dynamics simulations. The anisotropy of tryptophan and tyrosine fluorescence could be separated in three parts: (I) overall rotation of the protein (10(-9)s), (II) internal mobility of side chains (10(-10)s), and (III) a very fast relaxation (10(-12)s). We can only experimentally detect this very fast relaxation when the internal motion is not present. 相似文献
2.
Contribution of single tryptophan residues to the fluorescence and stability of ribonuclease Sa
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Alston RW Urbanikova L Sevcik J Lasagna M Reinhart GD Scholtz JM Pace CN 《Biophysical journal》2004,87(6):4036-4047
Ribonuclease Sa (RNase Sa) contains no tryptophan (Trp) residues. We have added single Trp residues to RNase Sa at sites where Trp is found in four other microbial ribonucleases, yielding the following variants of RNase Sa: Y52W, Y55W, T76W, and Y81W. We have determined crystal structures of T76W and Y81W at 1.1 and 1.0 A resolution, respectively. We have studied the fluorescence properties and stabilities of the four variants and compared them to wild-type RNase Sa and the other ribonucleases on which they were based. Our results should help others in selecting sites for adding Trp residues to proteins. The most interesting findings are: 1), Y52W is 2.9 kcal/mol less stable than RNase Sa and the fluorescence intensity emission maximum is blue-shifted to 309 nm. Only a Trp in azurin is blue-shifted to a greater extent (308 nm). This blue shift is considerably greater than observed for Trp71 in barnase, the Trp on which Y52W is based. 2), Y55W is 2.1 kcal/mol less stable than RNase Sa and the tryptophan fluorescence is almost completely quenched. In contrast, Trp59 in RNase T1, on which Y55W is based, has a 10-fold greater fluorescence emission intensity. 3), T76W is 0.7 kcal/mol more stable than RNase Sa, indicating that the Trp side chain has more favorable interactions with the protein than the threonine side chain. The fluorescence properties of folded Y76W are similar to those of the unfolded protein, showing that the tryptophan side chain in the folded protein is largely exposed to solvent. This is confirmed by the crystal structure of the T76W which shows that the side chain of the Trp is only approximately 7% buried. 4), Y81W is 0.4 kcal/mol less stable than RNase Sa. Based on the crystal structure of Y81W, the side chain of the Trp is 87% buried. Although all of the Trp side chains in the variants contribute to the unusual positive circular dichroism band observed near 235 nm for RNase Sa, the contribution is greatest for Y81W. 相似文献
3.
Margareta Sahlin Kyung-Bin Cho Stephan Pötsch Simon D. Lytton Yasmin Huque Michael R. Gunther Britt-Marie Sjöberg Ronald P. Mason Astrid Gräslund 《Journal of biological inorganic chemistry》2002,7(1-2):74-82
Catalytically important free radicals in enzymes are generally formed at highly specific sites, but the specificity is often lost in point mutants where crucial residues have been changed. Among the transient free radicals earlier found in the Y122F mutant of protein R2 in Escherichia coli ribonucleotide reductase after reconstitution with Fe2+ and O2, two were identified as tryptophan radicals. A third radical has an axially symmetric EPR spectrum, and is shown here using 17O exchange and simulations of EPR spectra to be a peroxyl adduct radical. Reconstitution of other mutants of protein R2 (i.e. Y122F/W48Y and Y122F/W107Y) implicates W48 as the origin of the peroxyl adduct. The results indicate that peroxyl radicals form on primary transient radicals on surface residues such as W48, which is accessible to oxygen. However, the specificity of the reaction is not absolute since the single mutant W48Y also gives rise to a peroxyl adduct radical. We used density functional calculations to investigate residue-specific effects on hyperfine coupling constants using models of tryptophan, tyrosine, glycine and cysteine. The results indicate that any peroxyl adduct radical attached to the first three amino acid alpha-carbons gives similar 17O hyperfine coupling constants. Structural arguments and experimental results favor W48 as the major site of peroxyl adducts in the mutant Y122F. Available molecular oxygen can be considered as a spin trap for surface-located protein free radicals. 相似文献
4.
Catalytically essential side-chain radicals have been recognized in a growing number of redox enzymes. Here we present a novel approach to study this class of redox cofactors. Our aim is to construct a de novo protein, a radical maquette, that will provide a protein framework in which to investigate how side-chain radicals are generated, controlled, and directed toward catalysis. A tryptophan and a tyrosine radical maquette, denoted alpha(3)W(1) and alpha(3)Y(1), respectively, have been synthesized. alpha(3)W(1) and alpha(3)Y(1) contain 65 residues each and have molecular masses of 7.4 kDa. The proteins differ only in residue 32, which is the position of their single aromatic side chain. Structural characterization reveals that the proteins fold in water solution into thermodynamically stable, alpha-helical conformations with well-defined tertiary structures. The proteins are resistant to pH changes and remain stable through the physiological pH range. The aromatic residues are shown to be located within the protein interior and shielded from the bulk phase, as designed. Differential pulse voltammetry was used to examine the reduction potentials of the aromatic side chains in alpha(3)W(1) and alpha(3)Y(1) and compare them to the potentials of tryptophan and tyrosine when dissolved in water. The tryptophan and tyrosine potentials were raised considerably when moved from a solution environment to a well-ordered protein milieu. We propose that the increase in reduction potential of the aromatic residues buried within the protein, relative to the solution potentials, is due to a lack of an effective protonic contact between the aromatic residues and the bulk solution. 相似文献
5.
Actin contains four tryptophan residues, W79, W86, W340, and W356, all located in subdomain 1 of the protein. Replacement of each of these residues with either tyrosine (W79Y and W356Y) or phenylalanine (W86F and W340F) generated viable proteins in the yeast Saccharomyces cerevisiae, which, when purified, allowed the analysis of the contribution of these residues to the overall tryptophan fluorescence of actin. The sum of the relative contributions of these tryptophans was found to account for the intrinsic fluorescence of wild-type actin, indicating that energy transfer between the tryptophans is not the main determinant of their quantum yield, and that these mutations induce little conformational change to the protein. This was borne out by virtually identical polymerization rates and similar myosin interactions of each of the mutants and the wild-type actin. In addition, these mutants allowed the dissection of the microenvironment of each tryptophan as actin undergoes conformational changes upon metal cation exchange and polymerization. Based on the relative tryptophan contributions determined from single mutants, a triple mutant of yeast actin (W79) was generated that showed small intrinsic fluorescence and should be useful for studies of actin interactions with actin-binding proteins. 相似文献
6.
M Vincent N Rouvière J Gallay 《Cellular and molecular biology, including cyto-enzymology》2000,46(6):1113-1131
The time-resolved fluorescence intensity and anisotropy decays of the immunophilin domain of FKBP59 (FKBP59-I)--a protein containing two tryptophan residues (the W89, buried in a hydrophobic pocket and the W59, water exposed)--were studied using the time-correlated single photon counting (TCSPC) technique. The synchrotron radiation machine Super-ACO (Orsay, France) was used as a pulsed light source (approximately 8MHz). A mainly dual and discrete excited state lifetime distribution was previously evidenced (Rouvière et al., 1997). The lifetime heterogeneity has been suggested to be relevant to the topological tryptophan heterogeneity. Indeed, taking into account the spectroscopic properties of the single tryptophan residue of the immunophilin FKBP12, a highly homologous protein containing a single tryptophan residue, the short- and the long-lived lifetime species were assumed to be related to the solvent-buried and to the solvent-exposed fluorescent residues, respectively. We definitely demonstrate this point by describing the dynamical properties of each tryptophan residue of the FKBP59-I as a function of the emission wavelength. The data of the polarized components of the fluorescence emission were analyzed by the Maximum Entropy Method using a one-dimensional model (each excited-state lifetime tau being associated with each rotational correlation time theta) and a two-dimensional model (without any a priori association constraint between the tau's and the theta's). The two dimensional analysis of the polarized fluorescence intensity decays by MEM show the existence of a correlation between fast picosecond dynamics of the indole ring with the shortest-lived and blue emitting species. Conversely, the long-lived and red emitting population is mainly associated to the Brownian motion of the protein. A protein flexibility of the region located around the W59 residue, but slightly contributing to the light depolarization process, is also evidenced and can be specifically attributed to the red emitting population. 相似文献
7.
Zelent B Odili S Buettger C Shiota C Grimsby J Taub R Magnuson MA Vanderkooi JM Matschinsky FM 《The Biochemical journal》2008,413(2):269-280
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. 相似文献
8.
D'Auria S Staiano M Varriale A Gonnelli M Marabotti A Rossi M Strambini GB 《Proteins》2008,71(2):743-750
Glutamine-binding protein (GlnBP) from Escherichia coli is a monomer (26 kDa) that is responsible for the first step in the active transport of L-glutamine across the cytoplasmic membrane. GlnBP consists of two domains (termed large and small) linked by two antiparallel beta-strands. The large domain is similar to the small domain but it contains two additional alpha-helices and three more short antiparallel beta-strands. The deep cleft formed between the two domains contains the ligand-binding site. The binding of L-glutamine leads to cleft closing and a significant structural change with the formation of the so-called "closed form" structure. The protein contains two tryptophan residues (W32 and W220) and 10 tyrosine residues. We used phosphorescence spectroscopy measurements to characterize the role of the two tryptophan residues in the protein structure in the absence and the presence of glutamine. Our results pointed out that the phosphorescence of GlnBP is easily detected in fluid solutions where the emission of the two tryptophan residues is readily discriminated by the drastic difference in the phosphorescence lifetime allowing the assignments of the short lifetime to W220 and the long lifetime to W32. In addition, our results showed that the triplet lifetime of the superficial W220 is unusually short because of intramolecular quenching by the proximal Y163. On the contrary, the lifetime of W32 is several hundred milliseconds long, implicating a well-ordered, compact fold of the surrounding polypeptide. The spectroscopic data were analyzed and discussed together with a detailed inspection of the 3D structure of GlnBP. 相似文献
9.
Resolution of the fluorescence decay of the two tryptophan residues of lac repressor using single tryptophan mutants. 总被引:4,自引:1,他引:3
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We have studied the time-resolved intrinsic tryptophan fluorescence of the lac repressor (a symmetric tetramer containing two tryptophan residues per monomer) and two single-tryptophan mutant repressors obtained by site-directed mutagenesis, lac W201Y and lac W220Y. These mutant repressor proteins have tyrosine substituted for tryptophan at positions 201 and 220, respectively, leaving a single tryptophan residue per monomeric subunit at position 220 for the W201Y mutant and at position 201 in the W220Y mutant. It was found that the two decay rates recovered from the analysis of the wild type data do not correspond to the rates recovered from the analysis of the decays of the mutant proteins. Each of these residues in the mutant repressors displays at least two decay rates. Global analysis of the multiwavelength data from all three proteins, however, yielded results consistent with the fluorescence decay of the wild type lac repressor corresponding simply to the weighted linear combination of the decays from the mutant proteins. The effect of ligation by the antagonistic ligands, inducer and operator DNA, was similar for all three proteins. The binding of the inducer sugar resulted in a quenching of the long-lived species, while binding by the operator decreased the lifetime of the short components. Investigation of the time-resolved anisotropy of the intrinsic tryptophan fluorescence in these three proteins revealed that the depolarization of fluorescence resulted from a fast motion and the global tumbling of the macromolecule. Results from the simultaneous global analysis of the frequency domain data sets from the three proteins revealed anisotropic rotations for the macromolecule, consistent with the known elongated shape of the repressor tetramer. In addition, it appears that the excited-state dipole of tryptophan 220 is alighed with the long axis of the repressor. 相似文献
10.
11.
The protein response to retinal chromophore isomerization in the visual pigment rhodopsin is studied using picosecond time-resolved UV resonance Raman spectroscopy. High signal-to-noise Raman spectra are obtained using a 1 kHz Ti:Sapphire laser apparatus that provides <3 ps visible (466 nm) pump and UV (233 nm) probe pulses. When there is no time delay between the pump and probe events, tryptophan modes W18, W16, and W3 exhibit decreased Raman scattering intensity. At longer pump-probe time delays of +5 and +20 ps, both tryptophan (W18, W16, W3, and W1) and tyrosine (Y1 + 2xY16a, Y7a, Y8a) peak intensities drop by up to 3%. These intensity changes are attributed to decreased hydrophobicity in the microenvironment near at least one tryptophan and one tyrosine residue that likely arise from weakened interaction with the beta-ionone ring of the chromophore following cis-to-trans isomerization. Examination of the crystal structure suggests that W265 and Y268 are responsible for these signals. These UV Raman spectral changes are nearly identical to those observed for the rhodopsin-to-Meta I transition, implying that impulsively driven protein motion by the isomerizing chromophore during the 200 fs primary transition drives key structural changes that lead to protein activation. 相似文献
12.
Halstead MF Ajtai K Penheiter AR Spencer JD Zheng Y Morrison EA Burghardt TP 《Biophysical journal》2007,93(10):3555-3566
The motor protein myosin binds actin and ATP, producing work by causing relative translation of the proteins while transducing ATP free energy. Smooth muscle myosin has one of four heavy chains encoded by the MYH11 gene that differ at the C-terminus and in the active site for ATPase due to alternate splicing. A seven-amino-acid active site insert in phasic muscle myosin is absent from the tonic isoform. Fluorescence increase in the nucleotide sensitive tryptophan (NST) accompanies nucleotide binding and hydrolysis in several myosin isoforms implying it results from a common origin within the motor. A wild-type tonic myosin (smA) construct of the enzymatic head domain (subfragment 1 or S1) has seven tryptophan residues and nucleotide-induced fluorescence enhancement like other myosins. Three smA mutants probe the molecular basis for the fluorescence enhancement. W506+ contains one tryptophan at position 506 homologous to the NST in other myosins. W506F has the native tryptophans except phenylalanine replaces W506, and W506+(Y499F) is W506+ with phenylalanine replacing Y499. W506+ lacks nucleotide-induced fluorescence enhancement probably eliminating W506 as the NST. W506F has impaired ATPase activity but retains nucleotide-induced fluorescence enhancement. Y499F replacement in W506+ partially rescues nucleotide sensitivity demonstrating the role of Y499 as an NST facilitator. The exceptional response of W506 to active site conformation opens the possibility that phasic and tonic isoforms differ in how influences from active site ATPase propagate through the protein network. 相似文献
13.
Picosecond protein dynamics of myoglobin in response to structural changes in heme upon CO dissociation were observed in a site-specific fashion for the first time using time-resolved UV resonance Raman spectroscopy. Transient UV resonance Raman spectra showed several phases of intensity changes in both tryptophan and tyrosine Raman bands. Five picoseconds after dissociation, the W18, W16, and W3 bands of tryptophan residues and the Y8a band of tyrosine residues decreased in intensity, followed by recovery of the Y8a band intensity in hundreds of picoseconds and recovery of the tryptophan bands in nanoseconds. These spectral changes suggest that the change in heme structure impulsively drives concerted movement of the EF helical section and that rearrangements toward a deoxy structure occur in the heme vicinity and in the A helix within a time frame of sub-nanoseconds to nanoseconds. 相似文献
14.
Muñiz VA Srinivasan S Boswell SA Meinhold DW Childs T Osuna R Colón W 《Protein science : a publication of the Protein Society》2011,20(2):302-312
Factor for inversion stimulation (FIS), a 98-residue homodimeric protein, does not contain tryptophan (Trp) residues but has four tyrosine (Tyr) residues located at positions 38, 51, 69, and 95. The equilibrium denaturation of a P61A mutant of FIS appears to occur via a three-state (N2 ⇆ I2 ⇆ 2U) process involving a dimeric intermediate (I2). Although it was suggested that this intermediate had a denatured C-terminus, direct evidence was lacking. Therefore, three FIS double mutants, P61A/Y38W, P61A/Y69W, and P61A/Y95W were made, and their denaturation was monitored by circular dichroism and Trp fluorescence. Surprisingly, the P61A/Y38W mutant best monitored the N2 ⇆ I2 transition, even though Trp38 is buried within the dimer removed from the C-terminus. In addition, although Trp69 is located on the protein surface, the P61A/Y69W FIS mutant exhibited clearly biphasic denaturation curves. In contrast, P61A/Y95W FIS was the least effective in decoupling the two transitions, exhibiting a monophasic fluorescence transition with modest concentration-dependence. When considering the local environment of the Trp residues and the effect of each mutation on protein stability, these results not only confirm that P61A FIS denatures via a dimeric intermediate involving a disrupted C-terminus but also suggest the occurrence of conformational changes near Tyr38. Thus, the P61A mutation appears to compromise the denaturation cooperativity of FIS by failing to propagate stability to those regions involved mostly in intramolecular interactions. Furthermore, our results highlight the challenge of anticipating the optimal location to engineer a Trp residue for investigating the denaturation mechanism of even small proteins. 相似文献
15.
Asparaginase II from Escherichia coli is well-known member of the bacterial class II amidohydrolases. Enzymes of this family utilize a peculiar catalytic mechanism in which a pair of threonine residues play pivotal roles. Another common feature is a mobile surface loop that closes over the active site when the substrates is bound. We have studied the motion of the loop by stopped-flow experiments using the fluorescence of tryptophan residues as the spectroscopic probe. With wild-type enzyme the fluorescence of the only tryptophan, W66, was monitored. Here asparagine induced a rapid closure of the loop. The rate constants of the process (100-150 s(-1) at 4 degrees C) were considerably higher than those of the rate-limiting catalytic step. A more selective spectroscopic probe was generated by replacing W66 with tyrosine and Y25, a component of the loop, with tryptophan. In the resulting enzyme variant, k(cat) and the rate of loop movement were reduced by factors of 10(2) and >10(3), respectively, while substrate binding was unaffected. This indicates that the presence of tyrosine in position 25 is essential for both loop closure and catalysis. Numerical simulations of the observed transients are consistent with a model where loop closure is an absolute prerequisite for substrate turnover. 相似文献
16.
Substitution of the conserved tryptophan 31 in Escherichia coli thioredoxin by site-directed mutagenesis and structure-function analysis 总被引:2,自引:0,他引:2
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. 相似文献
17.
Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps. The Y127W and Y159W mutant proteins have robust glycosylase activity toward DNA containing 1,N(6)-ethenoadenine (εA), within 4-fold of that of the wild-type enzyme, raising the possibility that tryptophan fluorescence could be used to probe the DNA binding and nucleotide flipping steps. Stopped-flow fluorescence was used to compare the time-dependent changes in tryptophan fluorescence and εA fluorescence. For both mutants, the tryptophan fluorescence exhibited two-step binding with essentially identical rate constants as were observed for the εA fluorescence changes. These results provide evidence that AAG forms an initial recognition complex in which the active site pocket is perturbed and the stacking of the damaged base is disrupted. Upon complete nucleotide flipping, there is further quenching of the tryptophan fluorescence with coincident quenching of the εA fluorescence. Although these mutations do not have large effects on the rate constant for excision of εA, there are dramatic effects on the rate constants for nucleotide flipping that result in 40-100-fold decreases in the flipping equilibrium relative to wild-type. Most of this effect is due to an increased rate of unflipping, but surprisingly the Y159W mutation causes a 5-fold increase in the rate constant for flipping. The large effect on the equilibrium for nucleotide flipping explains the greater deleterious effects that these mutations have on the glycosylase activity toward base lesions that are in more stable base pairs. 相似文献
18.
Several tryptophan (Trp) residues are conserved in G protein-coupled receptors (GPCRs). Relatively little is known about the contribution of these residues and especially of those in the fourth transmembrane domain in the function of the CB(2) cannabinoid receptor. Replacing W158 (very highly conserved in GPCRs) and W172 (conserved in CB(1) and CB(2) cannabinoid receptors but not in many other GPCRs) of the human CB(2) receptor with A or L or with F or Y produced different results. We found that the conservative change of W172 to F or Y retained cannabinoid binding and downstream signaling (inhibition of adenylyl cyclase), whereas removal of the aromatic side chain by mutating W172 to A or L eliminated agonist binding. W158 was even more sensitive to being mutated. We found that the conservative W158F mutation retained wild-type binding and signaling activities. However, W158Y and W158A mutants completely lost ligand binding capacity. Thus, the Trp side chains at positions 158 and 172 seem to have a critical, but different, role in cannabinoid binding to the human CB(2) receptor. 相似文献
19.
Phosphorescence and optically detected magnetic resonance (ODMR) measurements are reported on four single-tryptophan mutants of lac repressor protein from Escherichia coli: H74W/Wless, W201Y, Y273W/Wless, and F293W/Wless, where Wless represents a protein background containing the double mutation W201Y/W220Y. The single-tryptophan residues are located in the protein core region, either in the monomer-monomer interface of the tetrameric protein or in the region of the inducer binding cleft. Inducer binding elicits large changes in the energy (0,0-band wavelength shifts) and zero-field splitting energies (ZFS) of the triplet states for each of the mutant proteins except W201Y which exhibits more modest effects. F293W/Wless exists in two distinguishable conformations, only one of which appears to be sensitive to the presence of inducer. These effects of inducer binding can be attributed to a conformational change that alters specific polar interactions that occur at each affected tryptophan site. Changes in the tryptophan triplet state indicator depend on the existence of specific polar interactions that are altered by local atomic relocations. 相似文献
20.
Priyaranjan Pattanaik Gudihal Ravindra Chandana Sengupta Kapil Maithal Padmanabhan Balaram Hemalatha Balaram 《European journal of biochemistry》2003,270(4):745-756
Plasmodium falciparum triosephosphate isomerase (PfTIM) contains two tryptophan residues, W11 and W168. One is positioned in the interior of the protein, and the other is located on the active-site loop 6. Two single-tryptophan mutants, W11F and W168F, were constructed to evaluate the contributions of each chromophore to the fluorescence of the wild-type (wt) protein and to probe the utility of the residues as spectroscopic reporters. A comparative analysis of the fluorescence spectra of PfTIMwt and the two mutant proteins revealed that W168 possesses an unusual, blue-shifted emission (321 nm) and exhibits significant red-edge excitation shift of fluorescence. In contrast, W11 emits at 332 nm, displays no excitation dependence of fluorescence, and behaves like a normal buried chromophore. W168 has a much shorter mean lifetime (2.7 ns) than W11 (4.6 ns). The anomalous fluorescence properties of W168 are abolished on unfolding of the protein in guanidinium chloride (GdmCl) or at low pH. Analysis of the tryptophan environment using a 1.1-A crystal structure established that W168 is rigidly held by a complex network of polar interactions including a strong hydrogen bond from Y164 to the indole NH group. The environment is almost completely polar, suggesting that electrostatic effects determine the unusually low emission wavelength of W168. To our knowledge this is a unique observation of a blue-shifted emission from a tryptophan in a polar environment in the protein. The wild-type and mutant proteins show similar levels of enzymatic activity and secondary and tertiary structure. However, the W11F mutation appreciably destabilizes the protein to unfolding by urea and GdmCl. The fluorescence of W168 is shown to be extremely sensitive to binding of the inhibitor, 2-phosphoglycolic acid. 相似文献