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1.
Murine adenosine deaminase (mADA) is a 40 kDa (beta/alpha)(8)-barrel protein consisting of eight central beta-strands and eight peripheral alpha-helices containing four tryptophan residues. In this study, we investigated the urea-dependent behavior of the protein labeled with 6-fluorotryptophan (6-(19)F-Trp). The (19)F NMR spectrum of 6-(19)F-Trp-labeled mADA reveals four distinct resonances in the native state and three partly overlapped resonances in the unfolded state. The resonances were assigned unambiguously by site-directed mutagenesis. Equilibrium unfolding of 6-(19)F-Trp-labeled mADA was monitored using (19)F NMR based on these assignments. The changes in intensity of folded and unfolded resonances as a function of urea concentration show transition midpoints consistent with data observed by far-UV CD and fluorescence spectroscopy, indicating that conformational changes in mADA during urea unfolding can be followed by (19)F NMR. Chemical shifts of the (19)F resonances exhibited different changes between 1.0 and 6.0 M urea, indicating that local structures around 6-(19)F-Trp residues change differently. The urea-induced changes in local structure around four 6-(19)F-Trp residues of mADA were analyzed on the basis of the tertiary structure and chemical shifts of folded resonances. The results reveal that different local regions in mADA have different urea-dependent behavior, and that local regions of mADA change sequentially from native to intermediate topologies on the unfolding pathway. 相似文献
2.
19F NMR spectra of sodium fluoride in suspensions of human erythrocytes were seen to yield separate resonances for the F- populations inside and outside the cells. Selective saturation of the magnetization of the intracellular population gave rise to transfer of that saturation to the extracellular population. The extent of magnetization transfer was high and it was blocked by the capnophorin (band 3) anion exchange inhibitor 4,4-dini-trostilbene-2,2-disulfonic acid (DNDS). A series of magnetization-inversion transfer experiments was carried out for the range of intracellular fluoride concentrations of 11 mM to 136 mM and analysed using one-dimensional overdetermined exchange analysis. This yielded an estimate of the equilibrium exchange Michaelis constant and maximal velocity of 27 ± 3 mM and 180 ± 5 × 10-16 mol cell-1 s-1, respectively. There was no alteration of exchange flux of fluoride at an intracellular concentration of 49 mM in the presence of 50 mM glucose; thus suggesting no interaction between glucose and anions in capnophorin-mediated exchange of solutes. 相似文献
3.
Evolutionary conservation of domain-domain interactions 总被引:2,自引:1,他引:2
Background
Recently, there has been much interest in relating domain-domain interactions (DDIs) to protein-protein interactions (PPIs) and vice versa, in an attempt to understand the molecular basis of PPIs.Results
Here we map structurally derived DDIs onto the cellular PPI networks of different organisms and demonstrate that there is a catalog of domain pairs that is used to mediate various interactions in the cell. We show that these DDIs occur frequently in protein complexes and that homotypic interactions (of a domain with itself) are abundant. A comparison of the repertoires of DDIs in the networks of Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens shows that many DDIs are evolutionarily conserved.Conclusion
Our results indicate that different organisms use the same 'building blocks' for PPIs, suggesting that the functionality of many domain pairs in mediating protein interactions is maintained in evolution. 相似文献4.
R Wimmer T Herrmann M Solioz K Wüthrich 《The Journal of biological chemistry》1999,274(32):22597-22603
A recently discovered family of proteins that function as copper chaperones route copper to proteins that either require it for their function or are involved in its transport. In Enterococcus hirae the copper chaperone function is performed by the 8-kDa protein CopZ. This paper describes the NMR structure of apo-CopZ, obtained using uniformly (15)N-labeled CopZ overexpressed in Escherichia coli and NMR studies of the impact of Cu(I) binding on the CopZ structure. The protein has a betaalphabetabetaalphabeta fold, where the four beta-strands form an antiparallel twisted beta-sheet, and the two helices are located on the same side of the beta-sheet. A sequence motif GMXCXXC in the loop between the first beta-strand and the first alpha-helix contains the primary ligands, which bind copper(I). Binding of copper(I) caused major structural changes in this molecular region, as manifested by the fact that most NMR signals of the loop and the N-terminal part of the first helix were broadened beyond detection. This effect was strictly localized, because the remainder of the apo-CopZ structure was maintained after addition of Cu(I). NMR relaxation data showed a decreased correlation time of overall molecular tumbling for Cu(I)-CopZ when compared with apo-CopZ, indicating aggregation of Cu(I)-CopZ. The structure of CopZ is the first three-dimensional structure of a cupro-protein for which the metal ion is an exchangeable substrate rather than an integral part of the structure. Implications of the present structural work for the in vivo function of CopZ are discussed, whereby it is of special interest that the distribution of charged residues on the CopZ surface is highly uneven and suggests preferred recognition sites for other proteins that might be involved in copper transfer. 相似文献
5.
蛋白质折叠和分子伴侣 总被引:7,自引:1,他引:7
一个有活性的蛋白质分子不但有特定的氨基酸序列,还处于特定的由氨基酸序列决定的三维空间结构。三维结构的完整性受到干扰,生物活性也会发生变化:有时即使只是轻微的破坏,都可能导致其生物活性全部丧失。所以蛋白质的生物功能是与其三维空间结构密切联系在一起的。 相似文献
6.
Katarina Flemmer Karlsson Björn Walse Torbjörn Drakenberg Jan Kihlberg 《Letters in Peptide Science》1996,3(3):143-156
Summary Adhesive pili in uropathogenicE. coli are composed of a few different types of proteins which are assembled into the pilus by the chaperone PapD. Peptides from the C-terminus of these pilus proteins have been prepared by Fmoc solid-phase synthesis. Use of a polyethyleneglycol polystyrene resin was found to be essential for successful synthesis. The conformational propensities of the peptides were analyzed by CD and1H NMR spectroscopy, with special focus on PapG296-314 from the pilus adhesin which has previously showed the tightest binding to PapD. PapG296-314 was found to be flexible in different solutions, but with a significant propensity to adopt a -conformation. Interestingly the peptide is bound as a -strand in a crystalline complex with PapD. The peptides from three other pilus proteins could only be investigated in trifluoroethanol wher they displayed considerable -helicity in contrast to PapG296-314. These results suggest that conformational factors provide part of the explanation for the differential binding of pilus-related peptides to PapD.Abbreviations CD
circular dichroism
- CHAPS
3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate
- COSY
two-dimensional correlated spectroscopy
- DMF
dimethylformamide
- DMSO
dimethylsulfoxide
- FAB-MS
fast-atom-bombardment mass spectroscopy
- Fmoc
9-fluorenylmethoxycarbonyl
- Gal
galactose
- HOBt
1-hydroxybenzotriazole
- MeCN
acetonitrile
- MSNT
1-(2-mesitylenesulfonyl)-3-nitro-1,2,4-triazole
- NOE
nuclear Overhauser enhancement
- NOESY
two-dimensional nuclear Overhauser enhancement spectroscopy
- PEG-PS resin
polyethyleneglycol polystyrene resin
- ROESY
two-dimensional rotating frame nuclear Overhauser enhancement spectroscopy
- SDS
sodium dodecylsulfate
- TFA
trifluoroacetic acid
- TFE
2,2,2-trifluoroethanol
- TOCSY
two-dimensional total correlation spectroscopy
These authors have made equal contributions to the work presented.correspondence should be addressed. 相似文献
7.
M Scarpa P Viglino F Vianello A Rigo 《Biochemical and biophysical research communications》1991,174(1):163-168
F- added to an erythrocyte suspension shows two separated resonances arisen from intra and extracellular compartments. Cu, Zn superoxide dismutase dominates the longitudinal relaxation rate of the intracellular F- resonance, while diamagnetic interactions with hemoglobin contribute mainly to the transversal relaxation rate. 相似文献
8.
A 13C NMR study is reported of switch variant anti-dansyl antibodies, which possess the identical VH, VL, and CL domains in conjunction with highly homologous but not identical heavy-chain constant regions. Each of these antibodies has been selectively labeled with 13C at the carbonyl carbon of Trp, Tyr, His, or Cys residue by growing hybridoma cells in serum-free medium. Spectral assignments have been made by following the procedure described previously for the switch variant antibodies labeled with [1-13C]Met [Kato, K., Matsunaga, C., Igarashi, T., Kim, H., Odaka, A., Shimada, I., & Arata, Y. (1991) Biochemistry 30, 270-278]. On the basis of the spectral data collected for the antibodies and their proteolytic fragments, we discuss how 13C NMR spectroscopy can be used for the structural analyses of antigen binding and also of domain-domain interactions in the antibody molecule. 相似文献
9.
Background
Recently, there has been much interest in relating domain-domain interactions (DDIs) to protein-protein interactions (PPIs) and vice versa, in an attempt to understand the molecular basis of PPIs. 相似文献10.
Chen Chen Jun-Fei Zhao Qiang Huang Rui-Sheng Wang Xiang-Sun Zhang 《BMC systems biology》2012,6(Z1):S7
Background
As protein domains are functional and structural units of proteins, a large proportion of protein-protein interactions (PPIs) are achieved by domain-domain interactions (DDIs), many computational efforts have been made to identify DDIs from experimental PPIs since high throughput technologies have produced a large number of PPIs for different species. These methods can be separated into two categories: deterministic and probabilistic. In deterministic methods, parsimony assumption has been utilized. Parsimony principle has been widely used in computational biology as the evolution of the nature is considered as a continuous optimization process. In the context of identifying DDIs, parsimony methods try to find a minimal set of DDIs that can explain the observed PPIs. This category of methods are promising since they can be formulated and solved easily. Besides, researches have shown that they can detect specific DDIs, which is often hard for many probabilistic methods. We notice that existing methods just view PPI networks as simply assembled by single interactions, but there is now ample evidence that PPI networks should be considered in a global (systematic) point of view for it exhibits general properties of complex networks, such as 'scale-free' and 'small-world'.Results
In this work, we integrate this global point of view into the parsimony-based model. Particularly, prior knowledge is extracted from these global properties by plausible reasoning and then taken as input. We investigate the role of the added information extensively through numerical experiments. Results show that the proposed method has improved performance, which confirms the biological meanings of the extracted prior knowledge.Conclusions
This work provides us some clues for using these properties of complex networks in computational models and to some extent reveals the biological meanings underlying these general network properties.11.
Background
The development of high-throughput technologies has produced several large scale protein interaction data sets for multiple species, and significant efforts have been made to analyze the data sets in order to understand protein activities. Considering that the basic units of protein interactions are domain interactions, it is crucial to understand protein interactions at the level of the domains. The availability of many diverse biological data sets provides an opportunity to discover the underlying domain interactions within protein interactions through an integration of these biological data sets. 相似文献12.
We have incorporated 5-fluorouridine into several sites within a 19-mer RNA modelled on the translational operator of the MS2 bacteriophage. The 19F NMR spectra demonstrate the different chemical shifts of helical and loop fluorouridines of the hairpin secondary structure. Addition of salt gives rise to a species in which the loop fluorouridine gains the chemical shift of its helical counterparts, due to the formation of the alternative bi-molecular duplex form. This is supported by UV thermal melting behaviour which becomes highly dependent on the RNA concentration. Distinct 19F NMR signals for duplex and hairpin forms allow the duplex-hairpin equilibrium constant to be determined under a range of conditions, enabling thermodynamic characterisation and its salt dependence to be determined. Mg2+ also promotes duplex formation, but more strongly than Na+, such that at 25 degrees C, 10 mM MgCl2 has a comparable duplex-promoting effect to 300 mM NaCl. A similar effect is observed with Sr2+, but not Ca2+ or Ba2+. Additional hairpin species are observed in the presence of Na+ as well as Mg2+, Ca2+, Sr2+ and Ba2+ ions. The overall, ensemble average, hairpin conformation is therefore salt-dependent. Electrostatic considerations are thus involved in the balance between different hairpin conformers as well as the duplex-hairpin equilibrium. The data presented here demonstrate that 19F NMR is a powerful tool for the study of conformational heterogeneity in RNA, which is particularly important for probing the effects of metal ions on RNA structure. The thermodynamic characterisation of duplex-hairpin equilibria will also be valuable in the development of theoretical models of nucleic acid structure. 相似文献
13.
Fluorinated derivatives of the chelator o-aminophenol-N,N,O-triacetic acid (APTRA) have been developed, synthesized, and analyzed for use as 19F NMR indicators of free cytosolic magnesium concentration. Magnesium dissociation constants for the 4-fluoro, 5-fluoro, and 4-methyl-5-fluoro species were determined to be 3.1, 0.9, and 0.6 mM, respectively, on the basis of UV absorption measurements at 37 degrees C in 115 mM KCl and 20 mM NaCl, pH 7.1, buffered with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-tris-(hydroxymethyl)aminomethane. The corresponding pK values, which reflect protonation of the nitrogen atom, were determined by 19F NMR to be 4.15, 5.45, and 5.55, respectively, so that the chelators are insensitive to pH variations near the normal physiological range. The dissociation constants of these chelators for calcium ions are lower than those for magnesium but roughly 2-3 orders of magnitude above typical basal cytosolic free calcium levels, so that calcium ions will not interfere with the determinations of magnesium levels. 19F NMR studies carried out at 339.7 MHz indicate that magnesium ions are in slow exchange with the 5-fluoro and 4-methyl-5-fluoro APTRA derivatives and in fast exchange with the 4-fluoro APTRA derivative. In contrast, calcium ions were found to be in intermediate to fast exchange with all chelators. The apparent anomaly of higher thermodynamic stability of the APTRA complexes for calcium relative to magnesium but lower kinetic stability (higher k-1 values) for the calcium complexes reflects the very different association rates for the two ions. Thus, the magnesium association rates are 3 orders of magnitude slower than those for calcium ions.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
14.
Albert M Repetschnigg W Ortner J Gomes J Paul BJ Illaszewicz C Weber H Steiner W Dax K 《Carbohydrate research》2000,327(4):395-400
A fast method for the simultaneous detection of different glycosidolytic activities in commercially available enzyme preparations and crude culture filtrates was found in using, as substrate, a mixture of different glycosyl fluorides and 19F NMR spectroscopy as a screening technique. Accompanying studies regarding the hydrolytic stability of these fluorides in various buffer systems, as well as conditions of their long-term storage, were carried out. A simple procedure for the preparation of beta-D-mannopyranosyl fluoride in gram quantities is given. 相似文献
15.
Ionotropic glutamate receptors mediate the majority of vertebrate excitatory synaptic transmission. Although the structure of the GluR2 binding domain (S1S2) is well known (agonist binding site between two lobes), little is known about the time scales of conformational transitions or the relationship between dynamics and function. (19)F NMR ((19)F-labeled tryptophan) spectroscopy was used to monitor motions in the S1S2 domain bound to ligands with varying efficacy and in the apo state. One tryptophan (Trp-671) undergoes chemical exchange in some but not all agonists, consistent with mus-ms motion. The dynamics can be correlated to ligand affinity, and a likely source of the motion is a peptide bond capable of transiently forming hydrogen bonds across the lobe interface. Another tryptophan (Trp-767) appears to monitor motions of the relative positions of the lobes and suggests that the relative orientation in the apo- and antagonist-bound forms can exchange between at least two conformations on the ms time scale. 相似文献
16.
Vladimir S. Bondar Marelle G. Boersma Eugene L. Golovlev Jacques Vervoort Willem J.H. Van Berkel Zoya I. Finkelstein Inna P. Solyanikova Ludmila A. Golovleva Ivonne M.C.M. Rietjens 《Biodegradation》1998,9(6):475-486
Of all NMR observable isotopes 19F is the one perhaps most convenient for studies on biodegradation of environmental pollutants. The reasons underlying this potential of 19F NMR are discussed and illustrated on the basis of a study on the biodegradation of fluorophenols by four Rhodococcus strains. The results indicate marked differences between the biodegradation pathways of fluorophenols among the various Rhodococcus species. This holds not only for the level and nature of the fluorinated biodegradation pathway intermediates that accumulate, but also for the regioselectivity of the initial hydroxylation step. Several of the Rhodococcus species contain a phenol hydroxylase that catalyses the oxidative defluorination of ortho-fluorinated di- and trifluorophenols. Furthermore, it is illustrated how the 19F NMR technique can be used as a tool in the process of identification of an accumulated unknown metabolite, in this case most likely 5-fluoromaleylacetate. Altogether, the 19F NMR technique proved valid to obtain detailed information on the microbial biodegradation pathways of fluorinated organics, but also to provide information on the specificity of enzymes generally considered unstable and, for this reason, not much studied so far. 相似文献
17.
Interactive surface in the PapD chaperone cleft is conserved in pilus chaperone superfamily and essential in subunit recognition and assembly. 总被引:9,自引:0,他引:9
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The assembly of adhesive pili in Gram-negative bacteria is modulated by specialized periplasmic chaperone systems. PapD is the prototype member of the superfamily of periplasmic pilus chaperones. Previously, the alignment of chaperone sequences superimposed on the three dimensional structure of PapD revealed the presence of invariant, conserved and variable amino acids. Representative residues that protruded into the PapD cleft were targeted for site directed mutagenesis to investigate the pilus protein binding site of the chaperone. The ability of PapD to bind to fiber-forming pilus subunit proteins to prevent their participation in misassembly interactions depended on the invariant, solvent-exposed arginine-8 (R8) cleft residue. This residue was also essential for the interaction between PapD and a minor pilus adaptor protein. A mutation in the conserved methionine-172 (M172) cleft residue abolished PapD function when this mutant protein was expressed below a critical threshold level. In contrast, radical changes in the variable residue glutamic acid-167 (E167) had little or no effect on PapD function. These studies provide the first molecular details of how a periplasmic pilus chaperone binds to nascently translocated pilus subunits to guide their assembly into adhesive pili. 相似文献
18.
Kazuhito Tanabe Masaaki Sugiura Sei-ichi Nishimoto 《Bioorganic & medicinal chemistry》2010,18(18):6690-6694
We prepared oligodeoxynucleotides (ODNs) possessing a 5-fluorodeoxyuridine (5-FU) unit as a 19F-signal transmitter, and characterized their structures including single strand, duplex, and triplex using 19F NMR. The change in chemical shift induced by incorporation of 5-FU into the ODNs and the formation of higher order structures allowed monitoring of structural changes. Data from UV melting experiments and CD spectra were consistent with the spectral changes in the NMR studies. These 19F-labeled ODNs may be promising molecular probes for the identification of DNA structures in complicated biological conditions. 相似文献
19.
Domain structure and domain-domain interactions of recombinant tissue plasminogen activator 总被引:1,自引:0,他引:1
The melting of recombinant tissue plasminogen activator (rtPA) has been investigated by differential scanning calorimetry and fluorescence spectroscopy. At neutral pH, rtPA melts with only partial reversibility in a single sharp peak that can be deconvoluted into four transitions. By contrast, at acidic pH the melting process is spread over a broad range of temperature and is highly reversible. Under these conditions five transitions are resolved by deconvolution analysis. Additional measurements in 6 M guanidinium chloride reveal a sixth transition representing an extremely stable domain. Comparison of the melting curves of several fragments with those of the parent protein allowed all of the transitions to be assigned. The results indicate that rtPA is comprised of six independently folded domains. Two of these domains correspond to the two kringle modules whose thermodynamic properties are similar to those of the kringles in plasminogen. Two additional domains are formed by the epidermal growth factor (EGF)-like and finger modules, the latter of which is extremely stable, requiring the presence of a chemical denaturant for its melting to be observed. The serine protease module contains two more domains which at neutral pH melt cooperatively in a single transition but at low pH melt independently, accounting for the greater number of transitions observed there. Measurements with a 50-kDa fragment lacking the C-terminal half of the serine protease module and with a variant lacking the finger and EGF domains indicate that the serine protease domains interact strongly with and are stabilized by the finger and/or EGF domains in the intact protein. This interaction between domains located at opposite ends of the rtPA molecule produces a more compact structure. A better understanding of such interactions may enhance efforts to engineer plasminogen activators with improved thrombolytic properties. 相似文献
20.
Plasminogen activator inhibitor-1 (PAI-1) is a 43 kDa protein involved in the regulation of fibrinolysis. PAI-1 is the principal inhibitor of tissue-type plasminogen activator (t-PA), trapping the proteinase as an acyl-enzyme covalent complex (approximately 105 kDa). Four single tryptophan mutants of PAI-1 have been constructed in which three of the four tryptophan residues (Trp86, Trp139, Trp175, and Trp262) were replaced with phenylalanine. Biosynthetic incorporation of 5-fluorotryptophan (5F-Trp) into wild-type PAI-1 (5FW wtPAI-1) and the single tryptophan mutants (5FW86, 5FW139, 5FW175, and 5FW262) was achieved, allowing a (19)F NMR spectroscopic study of PAI-1 in its active and cleaved forms and in complex with t-PA. The (19)F NMR spectrum of active 5FW wtPAI-1 shows four clearly resolved peaks at -39.20, -49.26, -50.74, and -52.57 ppm relative to trifluoroacetic acid at 0 ppm. Unequivocal assignments of these four resonances in the spectrum of 5FW wtPAI-1 to specific tryptophan residues were accomplished by measuring the chemical shifts of the (19)F resonances of the single tryptophan mutants. There was close agreement between the resonances observed in 5FW wtPAI-1 and of those in the mutants for all three protein forms. This would imply little structural perturbation in the local structures of the tryptophan residues resulting from substitution by phenylalanine. The 5FW wtPAI-1 was observed to have lower second-order rate constant (k(app)) for the inhibition of t-PA than the natural tryptophan wtPAI-1, suggesting that the decreased activity may result from a small structural effect of the fluorine substituent of the indole ring. Further alterations in the k(app) and the stoichiometry of inhibition (SI) were observed in each of the mutants indicating an effect of the three tryptophan to phenylalanine mutations. Detailed interpretation of the (19)F NMR spectra of the PAI-1 mutants provides insights into the local segmental structure of the active form of the proteins and the structural changes that occur in the cleaved and t-PA complexed forms. 相似文献