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1.
Yokota A Takenaka H Oh T Noda Y Segawa S 《Protein science : a publication of the Protein Society》1998,7(8):1717-1727
Two peptide fragments from tuna cytochrome c (cyt c), N-fragment (residues 1-44 containing the heme) and C-fragment (residues 45-103), combine to form a 1:1 fragment complex. This was clearly proved by ion-spray mass spectrometry. It was found from CD and NMR spectra that the structure of the fragment complex formed is similar to that of an intact cyt c, although each isolated fragment itself is unstructured. Binding constants and enthalpies upon the complex formation were directly observed by isothermal titration calorimetry. Thermodynamic parameters (deltaG(o)b, deltaHb, deltaS(o)b, and deltaC(b)p)) associated with the complex formation were determined at various pHs and temperatures. DeltaHb was found to be almost independent of pH values. The change in heat capacity accompanying the complex formation (deltaC(b)p) was directly determined from the temperature dependence of deltaHb. In addition, the change in heat capacity and enthalpy upon tuna cyt c unfolding were determined by differential scanning calorimetry. Thermodynamic parameters for the unfolding/dissociation process of the fragment complex were compared with those for cyt c unfolding at pH 3.9 and 303 K. In a comparison of two unfolding processes, the heat capacity change of each was very close to the other, while both the unfolding enthalpy and entropy of the fragment complex were larger than those of tuna cyt c. These thermodynamic data suggest that the internal interactions between polar groups (hydrogen bonding) and nonpolar groups (van der Waals interactions) are preserved in the fragment complex as well as in the native state of cyt c. 相似文献
2.
Cytochrome c chimerae from natural and synthetic fragments: significance of the biological properties 总被引:4,自引:0,他引:4
Analogs of natural proteins have application in basic research, in medicine, and increasingly, in industry. Of the many methods developed for the fabrication of modified proteins, semisynthesis has so far been the most productive [R. E. Offord (1983) Proc. Eur. Peptide Symp., 17 31–42]. The technique of fragment-condensation semisynthesis takes as its basic raw material the native protein; fragments derived from it are manipulated by sequential degradation and resynthesis before recombination. While gene manipulation may overtake protein manipulation in general usefulness, certain types of analogs will remain accessible only by semisynthesis. Fragment condensation semisynthesis, as described above, has been used in preparing cytochrome c analogs [C. J. A. Wallace (1979) Proc. Am. Peptide Symp. 6 , 609–612]. To overcome the difficulty of replacing amino acids far from convenient cleavage sites, a number of variants of the method have also been used. These include fragment-specific chemical modification [C. J. A. Wallace & K. Rose (1983) Biochem. J. 215 , 651–658] and solid-phase synthesis of small [P. J. Boon, G. I. Tesser, H. H. K. Brinkhof & R. J. F. Nivard (1981) Proc. Eur. Peptide Symp. 16 , 127–130] or large fragments [L. E. Barstow, R. S. Young, E. Yakali, J. J. Sharp, J. C. O'Brien, P. W. Berman & H. A. Harbury (1977) Proc. Natl. Acad. Sci. USA 74 , 4148–4250]. The latter study gave low final recoveries, so we have prepared large peptides (38–42 residues) by classical solution methods. We have examined both their use in the reformation of the complete protein, and the physical and biochemical properties of the product analogs compared with those of the native proteins. 相似文献
3.
Reconstitution of a native-like SH2 domain from disordered peptide fragments examined by multidimensional heteronuclear NMR
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Ojennus DD Fleissner MR Wuttke DS 《Protein science : a publication of the Protein Society》2001,10(11):2162-2175
The N-terminal SH2 domain from the p85alpha subunit of phosphatidylinositol 3' kinase is cleaved specifically into 9- and 5-kD fragments by limited proteolytic digestion with trypsin. The noncovalent SH2 domain complex and its constituent tryptic peptides have been investigated using high-resolution heteronuclear magnetic resonance (NMR). These studies have established the viability of the SH2 domain as a fragment complementation system. The individual peptide fragments are predominantly unstructured in solution. In contrast, the noncovalent 9-kD + 5-kD complex shows a native-like (1)H-(15)N HSQC spectrum, demonstrating that the two fragments fold into a native-like structure on binding. Chemical shift analysis of the noncovalent complex compared to the native SH2 domain reveals that the highest degree of perturbation in the structure occurs at the cleavage site within a flexible loop and along the hydrophobic interface between the two peptide fragments. Mapping of these chemical shift changes on the structure of the domain reveals changes consistent with the reduction in affinity for the target peptide ligand observed in the noncovalent complex relative to the intact protein. The 5-kD fragment of the homologous Src protein is incapable of structurally complementing the p85 9-kD fragment, either in complex formation or in the context of the full-length protein. These high-resolution structural studies of the SH2 domain fragment complementation features establish the suitability of the system for further protein-folding and design studies. 相似文献
4.
Cytochrome c oxidase from bakers' yeast. I. Isolation and properties 总被引:35,自引:0,他引:35
T L Mason R O Poyton D C Wharton G Schatz 《The Journal of biological chemistry》1973,248(4):1346-1354
5.
NMR redox studies of Desulfovibrio vulgaris Cytochrome c3. Electron transfer mechanisms 总被引:1,自引:0,他引:1
J J Moura H Santos I Moura J LeGall G R Moore R J Williams A V Xavier 《European journal of biochemistry》1982,127(1):151-155
The 300-MHz proton NMR spectra of the tetrahaem cytochrome c3 from Desulfovibrio vulgaris were examined while varying the pH and the redox potential. The analysis of the complete NMR reoxidation pattern was done taking into account all the 16 redox states that can be present in the redox titration of a tetra-redox-center molecule. A network of saturation transfer experiments performed at different oxidation stages, between the fully reduced and the fully oxidized states, allowed the observation of different resonances for some of the haem methyl groups. In the present experimental conditions, some of the haems show a fast intramolecular electron exchange rate, but the intermolecular electron exchange is always slow. In intermediate reoxidation stages, large shifts of the resonances of some haem methyl groups were observed upon changing the pH. These shifts are discussed in terms of a pH dependence of the haem midpoint redox potentials. The physiological relevance of this pH dependence is discussed. 相似文献
6.
Cytochrome c interaction with membranes. Interaction of cytochrome c with isolated membrane fragments and purified enzymes 总被引:1,自引:0,他引:1
The midpoint redox potential of cytochrome c and the electron paramagnetic resonance spectra of nitroxide labeled cytochromes c were measured as a function of binding to purified cytochrome c oxidase, cytochrome c peroxidase, cytochrome b5 and succinate—cytochrome c reductase. The midpoint redox potential of horse heart cytochrome c is lowered in the presence of cytochrome c oxidase and succinate-cytochrome c reductase, but is unchanged in the presence of cytochrome c peroxidase or cytochrome b5. Further evidence of binding is afforded by an increase in correlation time, Tc, of the spin-labeled cytochrome c at methionine 65 upon binding to cytochrome c peroxidase, cytochrome c oxidase and succinate—cytochrome c reductase. The changes in midpoint redox potential and electron paramagnetic resonance spectrum of the spin-labeled derivative upon binding can either be the consequence of specific interaction leading to formation of ES complexes, or it can be due to nonspecific electrostatic interaction between positively charged groups on cytochrome c and negatively charged groups on the isolated cytochrome preparations. 相似文献
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9.
D Gopal G S Wilson R A Earl M A Cusanovich 《The Journal of biological chemistry》1988,263(24):11652-11656
The ion binding properties of horse, bovine, and tuna cytochrome c (both oxidized and reduced) have been measured using a combination of ultrafiltration, neutron activation, and ion chromatography. The ions investigated were chloride, phosphate, and Tris-cacodylate. Ion chromatography and neutron activation analysis techniques were employed to determine the concentration of free anions. Binding constants are obtained from modified Scatchard plots (in the range of 10-2000 M-1). The redox potentials for cytochrome c at different ionic strengths, pH 7.0, have been determined. In this paper we report the ionic strength and ion binding effects on the redox properties of horse, bovine, and tuna cytochrome c. Potential versus ionic strength dependence for horse, bovine, and tuna cytochrome c from the experimental data were compared with a theoretical model. 相似文献
10.
In earlier studies evidence was obtained for the existence of both high and low redox potential forms of cytochrome a3 (Hendler et al. 1986. Biophys. J. 49:717-729; Hendler and Sidhu. 1988. Biophys. J. 54:121-133). The current paper describes additional experiments that support this conclusion and then reviews a large number of experimental observations that appear to be consistent with the view that cytochrome a3 displays at least (see Sidhu and Hendler. 1990. Biophys. J. 57:1125-1140) two different forms, which are distinguishable by their redox potentials, spectra, and reactivity with CO. 相似文献
11.
Cytochrome c3 from the sulfate-reducing anaerobe Desulfovibrio africanus Benghazi: antigenic properties.
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Antisera were prepared against cytochromes c3 from Desulfovibrio africanus, D. vulgaris, and D. salexigens. Cross-reactions were observed between antisera to D. vulgaris and D. africanus cytochromes and heterologous cytochromes c3. A weak cross-reaction with antisera against both D. vulgaris and D. africanus cytochromes and the acid form of the D. salexigens cytochrome was seen; the basic form did not react. 相似文献
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13.
Cytochrome c from the fission yeast Schizosaccharomyces pombe has been purified. Its chromatographic and spectral properties are reported and compared to those of iso-1-cytochrome c from baker's yeast; the amino-acid composition is described. Schiz. pombe cytochrome c has a much lower affinity for Amberlite IRP64 than Sacch. cerevisiae iso-1-cytochrome c. Its alpha absorption band splits into three maxima (calpha1, calpha2, and calpha3) at -190 degrees C; this is unusual in yeasts, as shown by the low-temperature whole-cell absorption spectra which were examined in various yeast genera, species, and strains. A minor component can be separated by Amberlite chromatography. It exhibits the same low-temperature splitting of the alpha absorption band as the main fraction and it has a similar amino-acid composition with a notable exception: it is an unmethylated form of the cytochrome. 相似文献
14.
Ion channels from synaptic vesicle membrane fragments reconstituted into lipid bilayers. 总被引:1,自引:1,他引:0
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Cholinergic synaptic vesicles were isolated from the electric organ of Torpedo californica. Vesicle membrane proteins were reconstituted into planar lipid bilayers by the nystatin/ergosterol fusion technique. After fusion, a variety of ion channels were observed. Here we identify four channels and describe two of them in detail. The two channels share a conductance of 13 pS. The first is anion selective and strongly voltage dependent, with a 50% open probability at membrane potentials of -15 mV. The second channel is slightly cation selective and voltage independent. It has a high open probability and a subconductance state. A third channel has a conductance of 4-7 pS, similar to the subconductance state of the second channel. This channel is fairly nonselective and has gating kinetics different from those of the cation channel. Finally, an approximately 10-pS, slightly cation selective channel was also observed. The data indicate that there are one or two copies of each of the above channels in every synaptic vesicle, for a total of six channels per vesicle. These observations confirm the existence of ion channels in synaptic vesicle membranes. It is hypothesized that these channels are involved in vesicle recycling and filling. 相似文献
15.
Cytochrome c1 has been purified from mitochondria of the yeast Saccharomyces cerevisiae. The procedure involves solubilization withcholate, ammonium sulfate fractionation, disruption of the dytochrome b-c1 complex with mercaptoethanol and detergents, and chromatography on DEAE-cellulose. The final product is psectrally pure, contains up to 62 nmol of covalently bound heme per mg of protein and does not react with oxygen or carbon monoxide. Sodium dodecyl sulfate disaggregates the purified cytochrome into a single 31,000 dalton subunit carrying the covalently attached heme group. Many cytochrome c1 preparations contain in addition an 18,500 dalton polypeptide which is devoid of covalently bound heme. Since this polypeptide can be removed from the heme-carrying polypeptide by relatively mild procedures, it is probably not an essential subunit of cytochrome c1. Cytochrome c1 is extremely sensitive to proteolysis. If it si purified in the absence of protease inhibitors, a family of heme polypeptides with molecular weights of 29,000, 27,000, and 25,000 daltons is obtained. In the presence of the protease inhibitor phenylmethylsulfonylfluoride the purification yields predominantly a 31,000 dalton heme protein with only little contamination by a 29,000 dalton degradation product. In order to show that only the 31,000 dalton heme-polypeptide is the native species, yeast cells were labeled with the heme-precursor delta-amino[3H]levulinic acid, converted to protoplasts and directly lysed with dodecyl sulfate in the presence of protease inhibitors. Subsequent electrophoresis of the lysate in the presence of dodecyl sulfate reveals the covalently bound heme of cytochrome c1 as a single symmetrical peak at 31,000 daltons. 相似文献
16.
Cytochrome c3 from the sulfate-reducing anaerobe Desulfovibrio africanus Benghazi: purification and properties.
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Cytochrome c3 was purified from Desulfovibrio africanus Benghazi by extraction with alkaline deoxyribonuclease, fractionation with ammonium sulfate, batch elution from carboxymethyl Sephadex followed by chromatography on the same resin, and gel filtration on Sephadex G-75. The preparation was judge homogeneous by a variety of criteria. The molecular weight was determined in an analytical ultracentrifuge, and values between 14,400 and 15,490 were obtained, depending upon the presumed value of partial specific volume. Gel filtration on a calibrated column of Sephadex G-75 gave a value of 14,900 daltons. The amino acid composition was very similar to that observed for the cytochrome from other species of Desulfovibrio, with the exception of increased levels of ThR and PhE. S-Carboxymethylation of the protein before and after heme removal by HgCl2 demonstrated eight Cys molecules involved in heme binding or four heme sites per molecule. Titration with sodium dithionite under N2 gave an electrochemical potential (E' 0) of -276 mV relative to the normal hydrogen electrode. Electrochemical titration of the cytochrome gave a Nernst plot with two linear regions with E' 0 values of -0.376 and -0.534 V. The spectra produced at various potentials exhibited shifts in isosbestic points upon reduction, suggesting changes in conformation during the reaction. 相似文献
17.
Cytochrome c1 forms an active complex with cytochrome c as previously reported (Chiang, Y. L., Kaminsky, L. S., and King, T. E. (1976) J. Biol. Chem. 251, 29-36). It also forms a complex with cytochrome oxidase with heme ratio of 1:1. This cytochrome c1.oxidase complex has been purified by ammonium sulfate fractionation and is stable in media of high ionic strength (greater than 0.1 M) but dissociates as the pH deviates from neutral. The purified cytochrome c1 aggregates to an oligomer, presumably a pentamer. No agent has been found to depolymerize isolated c1 without denaturation. However, in the cytochrome c1.oxidase complex, these two cytochromes apparently were depolymerized to form smaller aggregates, if not monomeric units, as judged by sedimentation behavior. Cytochrome c1 also forms a ternary complex with cytochrome c and oxidase in the heme ratio of 1:1:1. This complex can be prepared by any of the following four methods: (i) c1 + c + oxidase: (ii) c1.c complex + oxidase; (iii) c1 + c.oxidase complex: or (iv) c + c1.oxidase complex. The mode of formation of these complexes is all from pure protein-protein interactions. Cytochrome c1 is also incorporated into phospholipid vesicles and these vesicles show about 200 molecules of phospholipid/cytochrome c1 in terms of heme. The spectrophotometric, circular dichroic, sedimentation behavior and enzymic properties of these complexes have been investigated. 相似文献
18.
Two fragments have been isolated and characterized from plasmin digests of human choriomammotropin. It was shown that these two fragments were derived from the cleavage of the Arg-Arg (positions 133-134) and the Lys-Gln (positions 140--141) bonds of the hormone: one has 133 amino acids and the other 51 amino acids. Both fragments were biologically and immunologically inactive. 相似文献
19.
The amino acid sequence of Schizosaccharomyces pombe cytochrome c has been established by automatic degradation of the protein and by manual degradation of fragments obtained by cyanogen bromide cleavage and chymotryptic digestion. The chymotryptic peptides were aligned by homology with other known cytochrome c sequences. The protein is 108 residues long, with a four-residue amino-terminal tail. It has only one methionine residue and differs from other fungal cytochromes c in lacking the one-residue deletion at the C-terminal end. After a cyanogen bromide step, an unexpected cleavage of the peptide chain before a cysteine residue was observed. This is ascribed to formation of a dehydroalanyl residue during an incomplete S-carboxymethylation of the apoprotein, and subsequent cleavage under acidic conditions. Experimental evidence is presented in favour of the proposed mechanisms. 相似文献
20.
L. Sawyer C.L. Jones A.M. Damas M.M. Harding R.O. Gould R.P. Ambler 《Journal of molecular biology》1981,153(3):831-835
The dihaem cytochrome c4 from Pseudomonas aeruginosa has been crystallized in space group P6522 with cell dimensions , and one molecule per asymmetric unit. Two heavy-atom derivatives, UO2(NO3)2 and K2Pt(NO2)4, which substitute at one and three sites, respectively, have allowed a low-resolution electron density map to be obtained. This shows clearly the two domains of the molecule. 相似文献