共查询到20条相似文献,搜索用时 31 毫秒
1.
C L Trevi?o W A Palmisano E R Birnbaum M T Henzl 《The Journal of biological chemistry》1990,265(17):9694-9700
The Eu3+ 7F0----5D0 excitation spectra of parvalbumin and oncomodulin are pH-dependent. Until now, it had been assumed that both the CD and EF ion-binding sites shared this property and that deprotonation of water molecules coordinated to the bound Eu3+ ions might be responsible for the pH dependence. Results obtained with the site-specific variant of oncomodulin known as D59E, in which glutamate replaces the aspartate naturally present at position 59, have necessitated substantial revision of these ideas. It now appears that the pH-dependent behavior is confined to the CD site. Moreover, we observe no corresponding change in the number of O-H oscillators coordinated to the bound Eu3+ ions in the pH range over which we observe the spectroscopic alteration. It is likely that the behavior results from deprotonation of one or more carboxyl groups clustered at the COOH-terminal end of the CD domain. 相似文献
2.
The 7F0----5D0 transition of Eu3+ was used to probe the metal-binding domains of rat oncomodulin and rat parvalbumin. Two distinct differences between the two proteins were observed. The first relates to the pH-dependent behavior of their 7F0----5D0 spectra, a phenomenon noted previously for other paravalbumins. In the case of rat parvalbumin, the spectral features associated with both metal-binding sites titrate concomitantly (pK alpha = 8.2); however, in the case of oncomodulin, the two sites titrate sequentially (pK alpha = 6.3 for the CD site; pK alpha = 8.3 for EF site). The proteins also contrast with regard to their discrimination for Eu3+ over Ca2+. The CD and EF sites in rat parvalbumin both display a large preference for Eu3+: (KCa/KEu)CD = 143 +/- 11 and (KCa/KEu)EF = 191 +/- 30. However, in the case of oncomodulin, although the EF site of oncomodulin greatly prefers the trivalent lanthanide ion (KCa/KEu = 300 +/- 80), the CD site exhibits a relatively minor preference (KCa/KEu = 11 +/- 1). 相似文献
3.
M T Henzl W D McCubbin C M Kay E R Birnbaum 《The Journal of biological chemistry》1985,260(14):8447-8455
Luminescence methods were used to examine the interaction of Eu(III) and Tb(III) with parvalbumin isozyme III from pike (Esox lucius). The bound lanthanide ions were excited both directly, via laser irradiation, and indirectly, via fluorescence energy transfer from adjacent phenylalanine residues. At high (175 microM) protein concentrations, the lanthanide titration curves exhibited pronounced quenching of luminescence at Ln3+:parvalbumin ratios above 2:1, in agreement with earlier reports (Donato, H., Jr., and Martin, R. B. (1974) Biochemistry 13, 4575-4579). However, in experiments performed with lower concentrations (10 microM), the titrations were well behaved and indicated a lanthanide:protein stoichiometry of 2:1. Equilibrium dialysis measurements performed with Eu(III) ruled out the existence of a third strong binding site which could cause the quenching of the luminescence at high protein concentrations. Similarly, careful analysis of the spectrum that results from direct excitation of the 7F0----5D0 transition of parvalbumin-bound Eu3+ ion revealed no peak attributable to a third Ln3+-binding site. The peak which has been construed by others (Rhee, M.-J., Sudnick, D. R., Arkle, V. K., and Horrocks, W. DeW., Jr. (1981) Biochemistry 20, 3328-3334) as evidence for a third site was shown to result from a pH-dependent spectral transition involving the europium ions bound at the CD and EF sites. Luminescent lifetime measurements performed on Tb(III)/parvalbumin solutions follow Stern-Volmer quenching kinetics at terbium:protein ratios in excess of 2:1, suggesting that the quenching results from collisional deactivation of the tightly bound ions by excess terbium ion free in solution. 相似文献
4.
The binding of Ca(II) and members of the trivalent lanthanide ion, Ln(III), series to apoparvalbumin (isotype pI = 4.75) from codfish (Gadus callarius L) results in the development of a distinctive sharp feature in the UV absorption spectrum at about 290 nm. Titration curves obtained by monitoring the spectral change in this region reveal a change in slope after the addition of 1 equiv of metal ion and no further rise after 2 equiv has been added, consistent with sequential binding to the principal EF and CD sites. Laser-induced luminescence excitation spectra of the 7F0----5D0 transition of bound Eu(III) demonstrate the quantitative binding of this ion to the principal sites and disclose the presence of a subsidiary site at pH values greater than 6. Metal ion competition experiments monitored by means of this excitation transition show that the early members of the Ln(III) ion series bind more tightly than those at the end. Tryptophan-sensitized Tb(III) luminescence reveals that this ion binds sequentially to the EF and CD sites, in that order. The intrinsic tryptophan fluorescence of apoparvalbumin is increased in a stepwise fashion as Ca(II) or Ln(III) ions bind sequentially, with the exceptions of Eu(III) and Yb(III). The binding of the latter two ions causes quenching of the protein fluorescence via an energy-transfer process which involves low-lying charge-transfer bands. The distance dependences of the tryptophan to Tb(III) and tryptophan to Eu(III) energy-transfer processes are observed to be identical, consistent with a F?rster-type mechanism in both cases. 相似文献
5.
Site-specific substitution of glutamate for aspartate at position 59 of rat oncomodulin 总被引:1,自引:0,他引:1
R C Hapak P J Lammers W A Palmisano E R Birnbaum M T Henzl 《The Journal of biological chemistry》1989,264(31):18751-18760
Replacement of the aspartate residue at position 59 of rat oncomodulin by glutamate by oligonucleotide-directed mutagenesis has afforded a protein which more closely resembles rat parvalbumin, at least judged by its interaction with the luminescent lanthanide ion Eu3+. The single-peak 7F0----5D0 spectrum observed at pH 5.0 with the fully bound wild-type protein is replaced by one which clearly shows two features at 5791 and 5796 A, arising from Eu3+ ions bound at the CD and EF sites, respectively. Furthermore, the pH dependence of the spectrum is substantially altered; the pKa observed for the CD domain, in which aspartate 59 residues, is shifted upward from pH 6.0 for the wild-type recombinant protein to pH 6.8 in the D59E mutant. Moreover, the maximum in the high-pH spectrum is shifted from 5781 to 5784 A. All three changes are indicative of a CD binding domain having increased parvalbumin-like character. Interestingly, however, the D59E substitution has only a modest effect on the Ca2+- and Mg2+-binding properties of the CD domain. For the wild-type protein, KCa = 7.8 x 10(-7) M and KMg = 3 x 10(-3) M. These affinities are more than an order of magnitude weaker than those seen for various parvalbumins and substantiate previous claims for calcium specificity made for the oncomodulin CD domain. Replacement of aspartate 59 by glutamate resulted in minor increases in affinity of the CD domain for Ca2+ (KCa = 5.5 x 10(-7) M) and Mg2+ (KMg = 1 x 10(-3) M). These findings strongly suggest that residues in oncomodulin besides aspartate 59 are important determinants of the observed calcium specificity of the CD calcium-binding domain. The consequences of the substitution at residue 59 appear to be confined to the CD domain. For the EF site in wild-type recombinant oncomodulin, KCa = 4.2 x 10(-8) M and KMg = 1.6 x 10(-4) M. The corresponding values for the D59E site-specific variant are identical within experimental error (KCa = 4.2 x 10(-8) M and KMg = 1.8 x 10(-4) M). 相似文献
6.
Excitation spectroscopy of the 7F0----5D0 transition of Eu3+ and diffusion-enhanced energy transfer are used to study metal-binding characteristics of the calcium-binding protein parvalbumin from codfish. Energy is transferred from Eu3+ ions occupying the CD- and EF-binding sites to the freely-diffusing Co(III) coordination complex energy acceptors: [Co(NH3)6]3+, [Co(NH3)5H2O]3+, [CoF(NH3)5]2+, [CoCl(NH3)5]2+, [Co(NO2)3(NH3)3], and [Co(ox)3]3-. In the absence of these inorganic energy acceptors, the excited-state lifetimes of Eu3+ bound to the CD and EF sites are indistinguishable, even in D2O; however, in the presence of the positively charged energy acceptor complexes, the Eu3+ probes in the cod parvalbumin have different excited-state lifetimes due to a greater energy-transfer site from Eu3+ in the CD site than from this ion in the EF site. The observation of distinct lifetimes for Eu3+ in the two sites allows the study of the relative binding site affinities and selectivity, using other members of the lanthanide ion series. Our results indicate that during the course of a titration of the metal-free protein, Eu3+ fills the two sites simultaneously. Eu3+ is competitively displaced by other Ln3+ ions, with the CD site showing a preference for the larger Ln3+ ions while the EF site shows little, if any, competitive selectivity across the Ln3+ ion series. 相似文献
7.
1. The kinetic properties of FoF1-ATPase from submitochondrial particles isolated from rat heart were studied, with emphasis to the pH effect. The velocity data were treated according to the Hill equation, and the results were discussed on the basis of the knowledge on the soluble F1-ATPase properties. 2. Three kinetic phases were observed in the range of pH 6.0-8.5, with apparent dissociation constant values (K0.5) of 0.001, 0.04 and 1.5 mM (respectively sites I, II and III) at pH 7.0. Their contribution to the total activity of the enzyme were pH-dependent on the range of 6.0-7.0, but not from 7.0 to 8.5, where the maximal velocity (V) for site III was some 4-fold larger than for site II, and the total V of sites II and III was some 40-fold larger than V assumed for site I. Therefore, two catalytic sites seem to participate significantly in the catalysis at steady-state condition. 3. Azide increased the sites II and III K0.5 values as well as decreased the site III V. In the presence of bicarbonate these two sites were not distinguishable, and the kinetic parameters at pH 7.0 were similar to those for sites II and III combined. Both azide and bicarbonate did not have a significant effect on site I, and this behavior was not pH-dependent. 4. The studies on the effect of pH on the kinetic parameters showed the following results: (1) the optimum pH for V was around 8.5; (2) decrease in the K0.5 values at pH below 7.0 for site II, and increase at pH over 7.0 for sites II and III; (3) in the pH range of 6.0-8.5 the Hill coefficient increased for site II, decreased for site III, and an intermediary effect was observed for the sites II and III combined, with a Michaelis-Menten behavior in the highest affinity pH, which was found in the physiological range. 相似文献
8.
Blundell CD Mahoney DJ Cordell MR Almond A Kahmann JD Perczel A Taylor JD Campbell ID Day AJ 《The Journal of biological chemistry》2007,282(17):12976-12988
TSG-6 is an inflammation-associated hyaluronan (HA)-binding protein that has anti-inflammatory and protective functions in arthritis and asthma as well as a critical role in mammalian ovulation. The interaction between TSG-6 and HA is pH-dependent, with a marked reduction in affinity on increasing the pH from 6.0 to 8.0. Here we have investigated the mechanism underlying this pH dependence using a combined approach of site-directed mutagenesis, NMR, isothermal titration calorimetry and microtiter plate assays. Analysis of single-site mutants of the TSG-6 Link module indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a histidine residue (His(4)) that is not within the HA-binding site. To understand this in molecular terms, the pH-dependent folding profile and the pK(a) values of charged residues within the Link module were determined using NMR. These data indicated that His(4) makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp(89)), whereas the other oxygen is simultaneously hydrogen-bonded to a key HA-binding residue (Tyr(12)). This molecular network transmits the change in ionization state of His(4) to the HA-binding site, which explains the loss of affinity at high pH. In contrast, simulations of the pH affinity curves indicate that another histidine residue, His(45), is largely responsible for the gain in affinity for HA between pH 3.5 and 6.0. The pH-dependent interaction of TSG-6 with HA (and other ligands) provides a means of differentially regulating the functional activity of this protein in different tissue microenvironments. 相似文献
9.
Oncomodulin, the parvalbumin-like calcium-binding protein frequently expressed in tumor tissue, was isolated from Morris hepatoma 5123tc and studied using the luminescent lanthanide ions, Eu3+ and Tb3+. Titrations of the apoprotein - whether monitored by indirect excitation of bound Tb3+, by direct laser excitation of bound Eu3+, or by quenching of the intrinsic tyrosine fluorescence - all indicated the presence of two high-affinity binding sites for lanthanide ions, as in parvalbumin. Moreover, the appearance of the Eu3+ 7F0----5D0 excitation spectrum of Eu2-oncomodulin was found to be highly pH-dependent, as previously observed with parvalbumin. At pH 5.0, it consists of a single peak centered at 5796 A, having a linewidth of approximately 6 A. At higher pH values, this spectrum is replaced by a broader, more symmetric peak at 5782 A. Oncomodulin, however, was found to differ from parvalbumin in at least one important respect: In contrast to the muscle-associated protein, the affinities of the CD site in oncomodulation for Tb3+ and Ca2+ were found to be rather similar, with KCa/KTb approximately equal to 11 +/- 2. 相似文献
10.
Human diferric transferrin was partially labeled with 59Fe at low or neutral pH (chemically labeled) and by replacement of diferric iron previously donated to rabbit reticulocytes (biologically labeled). Reticulocyte 59Fe uptake experiments with chemically labeled preparations indicated that iron bound at near neutral pH was more readily incorporated by reticulocytes than iron bound at low pH. The pH-dependent iron dissociation studies of biologically labeled transferrin solutions indicated that Fe3+, bound at the site from which the metal was initially utilized by the cells, dissociated between pH 5.8 and 7.4. In contrast, lower pH (5.2--5.8) was required to effect dissociation of iron that has remained bound to the protein after incubation with reticulocytes. These findings suggest that each human transferrin iron-binding site has different acid-base iron-binding properties which could be related to the observed heterogenic rabbit reticulocyte iron-donating properties of human transferrin and identifies that the near neutral iron-binding site initially surrenders its iron to these cells. 相似文献
11.
Type II restriction enzymes are homodimeric systems that bind four to eight base pair palindromic recognition sequences of DNA and catalyze metal ion-dependent phosphodiester cleavage. While Mg(II) is required for cleavage in these enzymes, in some systems Ca(II) promotes avid substrate binding and sequence discrimination. These properties make them useful model systems for understanding the roles of alkaline earth metal ions in nucleic acid processing. We have previously shown that two Ca(II) ions stimulate DNA binding by PvuII endonuclease and that the trivalent lanthanide ions Tb(III) and Eu(III) support subnanomolar DNA binding in this system. Here we capitalize on this behavior, employing a unique combination of luminescence spectroscopy and DNA binding assays to characterize Ln(III) binding behavior by this enzyme. Upon excitation of tyrosine residues, the emissions of both Tb(III) and Eu(III) are enhanced severalfold. This enhancement is reduced by the addition of a large excess of Ca(II), indicating that these ions bind in the active site. Poor enhancements and affinities in the presence of the active site variant E68A indicate that Glu68 is an important Ln(III) ligand, similar to that observed with Ca(II), Mg(II), and Mn(II). At low micromolar Eu(III) concentrations in the presence of enzyme (10-20 microM), Eu(III) excitation (7)F(0) --> (5)D(0) spectra yield one dominant peak at 579.2 nm. A second, smaller peak at 579.4 nm is apparent at high Eu(III) concentrations (150 microM). Titration data for both Tb(III) and Eu(III) fit well to a two-site model featuring a strong site (K(d) = 1-3 microM) and a much weaker site (K(d) approximately 100-200 microM). Experiments with the E68A variant indicate that the Glu68 side chain is not required for the binding of this second Ln(III) equivalent; however, the dramatic increase in DNA binding affinity around 100 microM Ln(III) for the wild-type enzyme and metal-enhanced substrate affinity for E68A are consistent with functional relevance for this weaker site. This discrimination of sites should make it possible to use lanthanide substitution and lanthanide spectroscopy to probe individual metal ion binding sites, thus adding an important tool to the study of restriction enzyme structure and function. 相似文献
12.
Members of the Hsp70 (heat-shock protein of 70 kDa) family of molecular chaperones bind to exposed hydrophobic stretches on substrate proteins in order to dissociate molecular complexes and prevent aggregation in the cell. Substrate affinity for the C-terminal domain of the Hsp70 is regulated by ATP binding to the N-terminal domain utilizing an allosteric mechanism. Our multi-dimensional NMR studies of a substrate-binding domain fragment (amino acids 387-552) from an Escherichia coli Hsp70, DnaK(387-552), have uncovered a pH-dependent conformational change, which we propose to be relevant for the full-length protein also. At pH 7, the C-terminus of DnaK(387-552) mimics substrate by binding to its own substrate-binding site, as has been observed previously for truncated Hsp70 constructs. At pH 5, the C-terminus is released from the binding site, such that DnaK is in the substrate-free state 10-20% of the time. We propose that the mechanism for the release of the tail is a loss of affinity for substrate at low pH. The pH-dependent fluorescence changes at a tryptophan residue near the substrate-binding pocket in full-length DnaK lead us to extend these conclusions to the full-length DnaK as well. In the context of the DnaK substrate-binding domain fragment, the release of the C-terminus from the substrate-binding site provides our first glimpse of the empty conformation of an Hsp70 substrate-binding domain containing a portion of the helical subdomain. 相似文献
13.
Coagulation factor IX-binding protein, isolated from Trimeresurus flavoviridis (IX-bp), is a C-type lectin-like protein. It is an anticoagulant consisting of homologous subunits, A and B. Each subunit has a Ca(2+)-binding site with a unique affinity (K(d) values of 14muM and 130muM at pH 7.5). These binding characteristics are pH-dependent and, under acidic conditions, the Ca(2+) binding of the low-affinity site was reduced considerably. In order to identify which site has high affinity and to investigate the pH-dependent Ca(2+) release mechanism, we have determined the crystal structures of IX-bp at pH 6.5 and pH 4.6 (apo form), and compared the Ca(2+)-binding sites with each other and with those of the solved structures under alkaline conditions; pH 7.8 and pH 8.0 (complexed form). At pH 6.5, Glu43 in the Ca(2+)-binding site of subunit A displayed two conformations. One (minor) is that in the alkaline state, and the other (major) is that at pH 4.6. However, the corresponding Gln43 residue of subunit B is in only a single conformation, which is almost identical with that in the alkaline state. At pH 4.6, Glu43 of subunit A adopts a conformation similar to that of the major conformer observed at pH 6.5, while Gln43 of subunit B assumes a new conformation, and both Ca(2+) positions are occupied by water molecules. These results showed that Glu43 of subunit A is much more sensitive to protonation than Gln43 of subunit B, and the conformational change of Glu43 occurs around pH6.5, which may correspond to the step of Ca(2+) release. 相似文献
14.
T Hofmann S Eng H Lilja T Drakenberg H J Vogel S Forsén 《European journal of biochemistry》1988,172(2):307-313
Europium(III) binding to 9-kDa calbindin from pig intestines was studied by direct excitation of the 7Fo----5Do transition of the ion and by near-ultraviolet circular dichroic spectroscopy. Europium(III) binding is clearly biphasic. As with other lanthanides the C-terminal metal-binding site (site II) is filled first. The europium ion in this site gives an excitation spectrum with a single peak at 579.1 nm (peak 2). The occupation of the N-terminal site (site I) by europium gives excitation spectra that are pH-dependent and show a peak at 579.4 nm (peak 1a) at pH 5 which shifts to 578.7 nm (peak 1b) over the pH range 5-7. At pH 8.07 the fluorescence from europium in site I largely disappears because of weak binding, whereas that from site II is quenched by about 75% in spite of full occupancy of the site as shown by circular dichroic titration. There is a strong interaction between the two sites in spite of the very different affinities. The fluorescence from site II increases stoichiometrically with the addition not only of the first equivalent of europium, but also concomitantly with the fluorescence from site I upon addition of the second equivalent. Furthermore, when Eu1-calbindin is titrated with calcium the fluorescence at 579.1 nm is quenched by about 30% during the addition of one equivalent of calcium which fills site I. Subsequent titration with large excesses of calcium displaces europium from site II. The affinity of site II for europium is about 100 times that of calcium under these conditions. 相似文献
15.
The macrophage scavenger receptor exhibits a pH-dependent conformational change around the carboxy-terminal half of the alpha-helical coiled coil domain, which has a representative amino acid sequence of a (defgabc)n heptad. We previously demonstrated that a peptide corresponding to this region formed a random coil structure at pH 7 and an alpha-helical coiled coil structure at pH 5 [Suzuki, K., Doi, T., Imanishi, T., Kodama, T., and Tanaka, T. (1997) Biochemistry 36, 15140-15146]. To determine the amino acid responsible for the conformational change, we prepared several peptides in which the acidic amino acids were replaced with neutral amino acids. Analyses of their structures by circular dichroism and sedimentation equilibrium gave the result that the presence of Glu242 at the d position was sufficient to induce the pH-dependent conformational change of the alpha-helical coiled coil domain. Furthermore, we substituted a Glu residue for the Ile residue at the d or a position of a de novo designed peptide (IEKKIEA)4, which forms a highly stable triple-stranded coiled coil. These peptides exhibited a pH-dependent conformational change similar to that of the scavenger receptor. Therefore, we conclude that a buried Glu residue in the hydrophobic core of a triple-stranded coiled coil has the potential to induce the pH-dependent conformational change. This finding makes it possible to elucidate the functions of natural proteins and to create a de novo protein designed to undergo a pH-dependent conformational change. 相似文献
16.
This is the first report of the redox potentials of glycolate oxidase. The pH dependence of the redox behavior as well as the effects of activators and inhibitors was studied. At pH 7.1 in 10 mM imidazole-chloride, Eo1' (EF1ox/EF1-.) is -0.033 +/- 0.010 V and Eo2' (EF1-./EF1redH-) is -0.017 +/- 0.017 V vs. the standard hydrogen electrode at 10 degrees C. A maximum of 29% flavin mononucleotide (FMN) anion radical is stabilized at half-reduction at pH 7.1 and 10 degrees C. Both redox couples of glycolate oxidase are pH-dependent from pH 7 to pH 9, and the FMN anion radical is stabilized in this range. The redox potentials of glycolate oxidase are shifted markedly positive of those of unbound FMN, consistent with the enzyme's function. The midpoint potential of glycolate oxidase is more positive than that of the glyoxalate/glycolate couple, and two-electron reduction of glycolate oxidase is thermodynamically favorable. The redox behavior of glycolate oxidase markedly contrasts that of other flavoprotein oxidases. For most flavoprotein oxidases, Eo1' is independent of pH from pH 7 to pH 9 and is much more positive than Eo2', which is pH-dependent. We present a mechanism that suggests a structural basis for the positive shifts and pH dependence of both Eo1' and Eo2' of glycolate oxidase. 相似文献
17.
Joseph V. Princiotto Edward J. Zapolski 《Biochimica et Biophysica Acta (BBA)/General Subjects》1976,428(3):766-771
Human diferric transferrin was partially labeled with 59Fe at low or neutral pH (chemically labeled) and by replacement of diferric iron previously donated to rabbit reticulocytes (biologically labeled). Reticulocyte 59 uptake experiments with chemically labeled preparations indicated that iron bound at near neutral ph was more readily incorporated by reticulocytes than iron bound at low pH. The pH-dependent iron dissociation studies of biologically labeled transferrin solutions indicated that Fe3+, bound at the site from which the metal was initially utilized by the cells, dissociated between pH 5.8 and 7.4. In contrast, lower pH (5.2–5.8) was required to effect dissociation of iron that had remained bound to the protein after incubation with reticulocytes. These findings suggest that each human transferrin iron-binding site has different acid-base iron-binding properties which could be related to the observed heterogenic rabbit reticulocyte iron-binding properties of human transferrin and identifies that the near neutral iron-donating site initially surrenders its iron to these cells. 相似文献
18.
Close coorelation of atomic absorption measurements for Ca(II) contents indicates that from pH 5.8-7.4 a twentyfold excess of EGTA1 removes but one of two Ca(II) from carp parvalbumin. Thus binding of the two Ca(II) appears to be noncooperative. The maximum in emission intensity observed at a nonintegral 1.4-1.7 equivs of added Tb(III) is shown to be due to quenching by excess Tb(III). The emission intensity at the maximum increased 40% upon dialysis to remove Tb(III) not bound in the CD or EF sites. Atomic absorption results show that both Ca(CD) and Ca(EF) of native parvalbumin are easily replaced by Tb(III). Emission of Tb(EF) is not quenched by Tb(CD), but by solution Tb(III) bound at a third site, perhaps the single water molecule bound to Tb(EF). Labeling of the single sulfhydryl group with a trifluoroacetonyl gorup yields a protein with ultraviolet circular dichroism, emission, and circularly polarized emission spectra closely similar to those of native parvalbumin. 相似文献
19.
Sebollela A Cagliari TC Limaverde GS Chapeaurouge A Sorgine MH Coelho-Sampaio T Ramos CH Ferreira ST 《The Journal of biological chemistry》2005,280(36):31949-31956
The biological activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) is modulated by the sulfated glycosaminoglycans (GAGs) heparan sulfate and heparin. However, the molecular mechanisms involved in such interactions are still not completely understood. We have proposed previously that helix C, one of the four alpha-helices of human GM-CSF (hGM-CSF), contains a GAG-binding site in which positively charged residues are spatially positioned for interaction with the sulfate moieties of the GAGs (Wettreich, A., Sebollela, A., Carvalho, M. A., Azevedo, S. P., Borojevic, R., Ferreira, S. T., and Coelho-Sampaio, T. (1999) J. Biol. Chem. 274, 31468-31475). Protonation of two histidine residues (His83 and His87) in helix C of hGM-CSF appears to act as a pH-dependent molecular switch to control the interaction with GAGs. Based on these findings, we have now generated a triple mutant form of murine GM-CSF (mGM-CSF) in which three noncharged residues in helix C of the murine factor (Tyr83, Gln85, and Tyr87) were replaced by the corresponding basic residues present in hGM-CSF (His83, Lys85, and His87). Binding assays on heparin-Sepharose showed that, at acidic pH, the triple mutant mGM-CSF binds to immobilized heparin with significantly higher affinity than wild type (WT) mGM-CSF and that neither protein binds to the column at neutral pH. The fact that even WT mGM-CSF binds to heparin at acidic pH indicates the existence of a distinct, lower affinity heparin-binding site in the protein. Chemical modification of the single histidine residue (His15) located in helix A of WT mGM-CSF with diethyl pyrocarbonate totally abolished binding to immobilized heparin. Moreover, replacement of His15 for an alanine residue significantly reduced the affinity of mGM-CSF for heparin at pH 5.0 and completely blocked heparin binding to a synthetic peptide corresponding to helix A of GM-CSF. These results indicate a major role of histidine residues in the regulation of the binding of GM-CSF to GAGs, supporting the notion that an acidic microenvironment is required for GM-CSF-dependent regulation of target cells. In addition, our results provide insight into the molecular basis of the strict species specificity of the biological activity of GM-CSF. 相似文献
20.
Two carboxylic acid side chains can, depending on their geometry and environment, share a proton in a hydrogen bond and form a carboxyl-carboxylate pair. In the Trichoderma reesei cellobiohydrolase Cel6A structure, five carboxyl-carboxylate pairs are observed. One of these pairs (D175-D221) is involved in catalysis, and three other pairs are found in, or close to the two surface loops covering the active site tunnel of the catalytic domain. To stabilize Cel6A at alkaline pH values, where deprotonation of the carboxylic acids leads to repulsion of their side chains, we designed two mutant enzymes. In the first mutant, one carboxyl-carboxylate pair (E107-E399) was replaced by a corresponding amide-carboxylate pair (Q107-E399), and in the second mutant, all three carboxyl-carboxylate pairs (E107-E399, D170-E184, and D366-D419) were mutated in a similar manner. The unfolding studies using both intrinsic tryptophan fluorescence and far-ultraviolet circular dichroism spectroscopy at different pH values demonstrate that the unfolding temperature (T(m)) of both mutants has changed, resulting in destabilization of the mutant enzymes at acidic pH and stabilization at alkaline pH. The effect of stabilization seems additive, as a Cel6A triple mutant is the most stable enzyme variant. This increased stability is also reflected in the 2- or 4-fold increased half-life of the two mutants at alkaline pH, while the catalytic rate on cellotetraose (at t = 0) has not changed. Increased operational stability at alkaline pH was also observed on insoluble cellulosic substrates. Local conformational changes are suggested to take place in the active site loops of Cel6A wild-type enzyme at elevated pHs (pH 7), affecting to the end-product spectrum on insoluble cellulose. The triple mutant does not show such pH-dependent behavior. Overall, our results demonstrate that carboxyl-carboxylate pair engineering is a useful tool to alter pH-dependent protein behavior. 相似文献