| 1. |
- Ademark, Pia, et al.
(författare)
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Softwood hemicellulose-degrading enzymes from Aspergillus niger: Purification and properties of a β-mannanase
- 1998
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Ingår i: Journal of Biotechnology. - 1873-4863. ; 63:3, s. 199-210
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Tidskriftsartikel (refereegranskat)abstract
- The enzymes needed for galactomannan hydrolysis, i.e. β-mannanase, α-galactosidase and β-mannosidase, were produced by the filamentous fungus Aspergillus niger. The β-mannanase was purified to electrophoretic homogeneity in three steps using ammonium sulfate precipitation, anion-exchange chromatography and gel filtration. The purified enzyme had an isoelectric point of 3.7 and a molecular mass of 40 kDa. Ivory nut mannan was degraded mainly to mannobiose and mannotriose when incubated with the β-mannanase. Analysis by 1H NMR spectroscopy during hydrolysis of mannopentaose showed that the enzyme acts by the retaining mechanism. The N-terminus of the purified A. niger β-mannanase was sequenced by Edman degradation, and comparison with Aspergillus aculeatus β-mannanase indicated high identity. The enzyme most probably lacks a cellulose binding domain since it was unable to adsorb on cellulose.
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| 2. |
- Anderson, Lars, et al.
(författare)
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Kinetics and stereochemistry of the Cellulomonas fimi beta-mannanase studied using H-1-NMR
- 2008
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Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 26:1-2, s. 86-95
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Tidskriftsartikel (refereegranskat)abstract
- Endo-1,4-beta-mannanases (beta-mannanases) randomly hydrolyse the mannosidic bonds within the main chain of various mannans and heteromannans. Some of these polysaccharides are hemicelluloses, a major part of the plant cell-wall. The beta-mannanases have been assigned to family 5 and 26 of the glycoside hydrolase clan A. This work presents a detailed kinetic analysis of the family 26 beta-mannanase CfMan26A from the soil-bacterium Cellulomonas fimi. The full-length enzyme consists of five modules: a family 26 catalytic module, an immunoglobulin-like module, a mannan-binding module, a surface layer homology-module and a module of unknown function. A truncated variant consisting of the catalytic module and the immunoglobulin-like module was used in these studies. The degradation of mannotriose, mannotetraose and mannopentaose was studied by H-1-NMR. First, the mutarotation of one of the hydrolysis products (mannose) was determined to be 1.7 10(-5) s(-1) at 5 degrees C and pH 5.0. As expected for a family 26 glycoside hydrolase, the hydrolysis was shown to proceed with overall retention of the anomeric configuration. Many 'retaining' enzymes can perform transglycosylation reactions. However, no transglycosylation could be detected. Kinetic constants were calculated from progress curves using computer simulation. It was revealed that the -3 subsite had a greater impact on the apparent k(cat)/K-m ratio (the catalytic efficiency) than the +2 subsite. The beta-anomer of mannotriose was hydrolysed 1000-times more efficiently than the alpha-anomer indicating selectivity for the beta- over the alpha-anomer in the +1 subsite. With background information from the previous published 3D-structure of the truncated variant of Man26A, a structural explanation for the observations is discussed.
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| 3. |
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| 4. |
- André, Ingemar, et al.
(författare)
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Streptococcal M protein: Structural studies of the hypervariable region, free and bound to human C4BP
- 2006
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 45:14, s. 4559-4568
-
Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pyogenes is a Gram-positive bacterium that causes several diseases, including acute tonsillitis and toxic shock syndrome. The surface-localized M protein, which is the most extensively studied virulence factor of S. pyogenes, has an similar to 50-residue N-terminal hypervariable region (HVR) that plays a key role in the escape of the host immunity. Despite the extensive sequence variability in this region, many HVRs specifically bind human C4b-binding protein (C4BP), a plasma protein that inhibits complement activation. Although the more conserved parts of M protein are known to have dimeric coiled-coil structure, it is unclear whether the HVR also is a coiled coil. Here, we use nuclear magnetic resonance (NMR) to study the conformational properties of HVRs from M4 and M22 proteins in isolation and in complex with the M protein binding portion of C4BP. We conclude that the HVRs of M4 and M22 are folded as coiled coils and that the folded nucleus of the M4 HVR has a length of similar to 27 residues. Moreover, we demonstrate that the C4BP binding surface of M4-N is found within a region of four heptad repeats. Using molecular modeling, we propose a model for the structure of the M4 HVR that is consistent with our experimental information from NMR spectroscopy.
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| 5. |
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| 6. |
- Drakenberg, Torbjörn, et al.
(författare)
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Calcium in Mammalian Cells
- 2006
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Ingår i: Biological Inorganic Chemistry: Structure and Reactivity. - 9781891389436 ; , s. 635-646
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 7. |
- Drakenberg, Torbjörn, et al.
(författare)
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Calcium Ion Binding to Pancreatic Phospholipase A2 and Its Zymogen : A 43Ca NMR Study
- 1984
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 23:11, s. 2387-2392
-
Tidskriftsartikel (refereegranskat)abstract
- Calcium ion binding to phospholipase A2 and its zymogen has been studied by 43Ca NMR. The temperature dependence of the band shape of the calcium-43 NMR signal has been used to calculate the calcium ion exchange rate. The on-rate was calculated to be 5 × 106 M-1 s-1, which is 2 orders of magnitude less than the diffusion limit of the hydrated Ca2+ ion in water. The 43Ca quadrupole coupling constant for calcium ions bound to phospholipase, χ = 1.4 MHz, is significantly larger than those found for EF-hand proteins, indicating a less symmetric site. For prophospholipase A2, we found χ = 0.8 MHz, indicating a calcium binding site, which is somewhat more symmetric than the EF-hand sites. The dependence of the Ca NMR band shape on the calcium ion concentration showed that there are two cation binding sites on the phospholipase A2 molecule: K1 = 4 × 103 M-1 and K2 = 20 M. The strong site was found to be affected by a pKa = 6.5 and the weak site by pKa = 4.5.
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| 8. |
- Drakenberg, Torbjörn, et al.
(författare)
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Solution Structure of the Ca(2+)-Binding EGF3-4 Pair from Vitamin K-Dependent Protein S: Identification of an Unusual Fold in EGF3(,).
- 2005
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 44:24, s. 8782-8789
-
Tidskriftsartikel (refereegranskat)abstract
- Vitamin K-dependent protein S is a cofactor of activated protein C, a serine protease that regulates blood coagulation. Deficiency of protein S can cause venous thrombosis. Protein S has four EGF domains in tandem; domains 2-4 bind calcium with high affinity whereas domains 1-2 mediate interaction with activated protein C. We have now solved the solution structure of the EGF3-4 fragment of protein S. The linker between the two domains is similar to what has been observed in other calcium-binding EGF domains where it provides an extended conformation. Interestingly, a disagreement between NOE and RDC data revealed a conformational heterogeneity within EGF3 due to a hinge-like motion around Glu186 in the Cys-Glu-Cys sequence, the only point in the domain where flexibility is allowed. The dominant, bent conformation of EGF3 in the pair has no precedent among calcium-binding EGF domains. It is characterized by a change in the angle of Glu186 from 160 ± 40, as seen in ten other EGF domains, to 0 ± 15. NOESY data suggest that Tyr193, a residue not conserved in other calcium-binding EGF domains (except in the homologue Gas6), induces the unique fold of EGF3. However, SAXS data, obtained on EGF1-4 and EGF2-4, showed a dominant, extended conformation in these fragments. This may be due to a counterproductive domain-domain interaction between EGF2 and EGF4 if EGF3 is in a bent conformation. We speculate that the ability of EGF3 to adopt different conformations may be of functional significance in protein-protein interactions involving protein S.
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| 9. |
- Erb, Eva-Maria, et al.
(författare)
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Interaction of bovine coagulation factor X and its glutamic-acid-containing fragments with phospholipid membranes. A surface plasmon resonance study.
- 2002
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 269:12, s. 3041-3046
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Tidskriftsartikel (refereegranskat)abstract
- The interaction of blood coagulation factor X and its Gla-containing fragments with negatively charged phospholipid membranes composed of 25 mol% phosphatidylserine (PtdSer) and 75 mol% phosphatidylcholine (PtdCho) was studied by surface plasmon resonance. The binding to 100 mol% PtdCho membranes was negligible. The calcium dependence in the membrane binding was evaluated for intact bovine factor X (factor X) and the fragment containing the Gla-domain and the N-terminal EGF (epidermal growth factor)-like domain, Gla-EGFN, from factor X. Both proteins show the same calcium dependence in the membrane binding. Calcium binding is cooperative and half-maximum binding was observed at 1.5 mm and 1.4 mm, with the best fit to the experimental data with three cooperatively bound calcium ions for both the intact protein and the fragment. The dissociation constant (Kd) for binding to membranes containing 25 mol% PtdSer decreased from 4.6 microm for the isolated Gla-domain to 1 microm for the fragments Gla-EGFN and Gla-EGFNC (the Gla-domain and both EGF-like domains) fragments and to 40 nm for the entire protein as zymogen, activated enzyme or in the active-site inhibited form. Analysis of the kinetics of adsorption and desorption confirmed the equilibrium binding data.
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| 10. |
- Frick, Inga-Maria, et al.
(författare)
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Convergent evolution among immunoglobulin G-binding bacterial proteins
- 1992
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Ingår i: Proceedings of the National Academy of Sciences. - 1091-6490. ; 89:18, s. 8532-8536
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Tidskriftsartikel (refereegranskat)abstract
- Protein G, a bacterial cell-wall protein with high affinity for the constant region of IgG (IgGFc) antibodies, contains homologous repeats responsible for the interaction with IgGFc. A synthetic peptide corresponding to an 11-amino acid-long sequence in the COOH-terminal region of the repeats was found to bind to IgGFc and block the interaction with protein G. Moreover, two other IgGFc-binding bacterial proteins (proteins A and H), which do not contain any sequences homologous to the peptide, were also inhibited in their interactions with IgGFc by the peptide. Finally, a decapeptide based on a sequence in IgGFc blocked the binding of all three proteins to IgGFc. This unusually clear example of convergent evolution emphasizes the complexity of protein-protein interactions and suggests that bacterial surface-protein interaction with host protein adds selective advantages to the microorganism.
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| 11. |
- Ghasriani, Houman, et al.
(författare)
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A model of the complex between human beta-microseminoprotein and CRISP-3 based on NMR data.
- 2009
-
Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 378:2, s. 235-239
-
Tidskriftsartikel (refereegranskat)abstract
- beta-Microseminoprotein (MSP), a 10kDa seminal plasma protein, forms a tight complex with cysteine-rich secretory protein 3 (CRISP-3) from granulocytes. The 3D structure of human MSP has been determined but there is as yet no 3D structure for CRISP-3. We have now studied the complex between human MSP and CRISP-3 with multidimensional NMR. (15)N-HSQC spectra show substantial differences between free and complexed hMSP. Using several 3D-NMR spectra of triply labeled hMSP in complex with a recombinant N-terminal domain of CRISP-3, most of the backbone of hMSP could be assigned. The data show that only one side of hMSP, comprising beta-strands 1, 4, 5, and 8 are affected by the complex formation, indicating that beta-strands 1 and 8 form the main binding surface. Based on this we present a tentative structure for the hMSP-CRISP-3 complex using the known crystal structure of triflin as a model of CRISP-3.
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| 12. |
- Ghasriani, Houman, et al.
(författare)
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Solution structures of human and porcine beta-microseminoprotein
- 2006
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 362:3, s. 502-515
-
Tidskriftsartikel (refereegranskat)abstract
- beta-Microseminoprotein (MSP) is a small cysteine-rich protein (molecular mass about 10 kDa) first isolated from human seminal plasma and later identified in several other organisms. The function of MSP is not known, but a recent study has shown MSP to bind CRISP-3, a protein present in neutrophilic granulocytes. The amino acid sequence is highly variable between species raising the question of the evolutionary conservation of the 3D structure. Here we present NMR solution structures of both the human and the porcine MSP. The two proteins (sequence identity 51%) have a very similar 3D structure with the secondary structure elements well conserved and with most of the amino acid substitutions causing a change of charge localized to one side of the molecule. MSP is a beta-sheet-rich protein with two distinct domains. The N-terminal domain is composed of a four-stranded beta-sheet, with the strands arranged according to the Greek key-motif, and a less structured part. The C-terminal domain contains two two-stranded beta-sheets with no resemblance to known structural motifs. The two domains, connected to each other by the peptide backbone, one disulfide bond, and interactions between the N and C termini, are oriented to give the molecule a rather extended structure. This global fold differs markedly from that of a previously published structure for porcine MSP, in which the two domains have an entirely different orientation to each other. The difference probably stems from a misinterpretation of ten specific inter-domain NOEs. (c) 2006 Elsevier Ltd. All rights reserved.
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| 13. |
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| 14. |
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| 15. |
- Gorecki, Kamil, et al.
(författare)
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The Na(+) transport in Gram-positive bacteria defect in the Mrp antiporter complex measured with (23)Na-NMR.
- 2014
-
Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 445:Online 15 October 2013, s. 80-86
-
Tidskriftsartikel (refereegranskat)abstract
- (23)Na-NMR has previously been used to monitor Na(+) translocation across membranes in Gram-negative bacteria and in various other organelles and liposomes using a membrane-impermeable shift reagent to resolve the signals resulting from internal and external Na(+). In this work, the (23)Na-NMR method was adapted for measurements of internal Na(+) concentration in the Gram-positive bacterium Bacillus subtilis, with the aim of assessing the Na(+) translocation activity of the Mrp antiporter complex, a member of the Cation Proton Antiporter-3 (CPA-3) family. The sodium sensitive growth phenotype observed in a B. subtilis strain with the gene encoding MrpA deleted, could indeed be correlated to the inability of this strain to maintain a lower internal than external Na(+) concentration.
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| 16. |
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| 17. |
- Hellstrand, Erik, et al.
(författare)
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Complete high-density lipoproteins in nanoparticle corona.
- 2009
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 276:12, s. 3372-3381
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Tidskriftsartikel (refereegranskat)abstract
- In a biological environment, nanoparticles immediately become covered by an evolving corona of biomolecules, which gives a biological identity to the nanoparticle and determines its biological impact and fate. Previous efforts at describing the corona have concerned only its protein content. Here, for the first time, we show, using size exclusion chromatography, NMR, and pull-down experiments, that copolymer nanoparticles bind cholesterol, triglycerides and phospholipids from human plasma, and that the binding reaches saturation. The lipid and protein binding patterns correspond closely with the composition of high-density lipoprotein (HDL). By using fractionated lipoproteins, we show that HDL binds to copolymer nanoparticles with much higher specificity than other lipoproteins, probably mediated by apolipoprotein A-I. Together with the previously identified protein binding patterns in the corona, our results imply that copolymer nanoparticles bind complete HDL complexes, and may be recognized by living systems as HDL complexes, opening up these transport pathways to nanoparticles. Apolipoproteins have been identified as binding to many other nanoparticles, suggesting that lipid and lipoprotein binding is a general feature of nanoparticles under physiological conditions.
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| 18. |
- Hsiao, Ya-Wen, et al.
(författare)
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NMR structure determination of proteins supplemented by quantum chemical calculations: Detailed structure of the Ca2+ sites in the EGF34 fragment of protein S
- 2005
-
Ingår i: Journal of Biomolecular NMR. - : Springer Science and Business Media LLC. - 1573-5001 .- 0925-2738. ; 31:2, s. 97-114
-
Tidskriftsartikel (refereegranskat)abstract
- We present and test two methods to use quantum chemical calculations to improve standard protein structure refinement by molecular dynamics simulations restrained to experimental NMR data. In the first, we replace the molecular mechanics force field ( employed in standard refinement to supplement experimental data) for a site of interest by quantum chemical calculations. This way, we obtain an accurate description of the site, even if a molecular mechanics force field does not exist for this site, or if there is little experimental information about the site. Moreover, the site may change its bonding during the refinement, which often is the case for metal sites. The second method is to extract a molecular mechanics potential for the site of interest from a quantum chemical geometry optimisation and frequency calculation. We apply both methods to the two Ca2+ sites in the epidermal growth factor-like domains 3 and 4 in the vitamin K-dependent protein S and compare them to various methods to treat these sites in standard refinement. We show that both methods perform well and have their advantages and disadvantages. We also show that the glutamate Ca2+ ligand is unlikely to bind in a bidentate mode, in contrast to the crystal structure of an EGF domain of factor IX.
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| 19. |
- Johansson, Charlotta, et al.
(författare)
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The Solution Structures of Mutant Calbindin D9k's, As Determined by NMR, Show That the Calcium-Binding Site Can Adopt Different Folds
- 1993
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 32:33, s. 8429-8438
-
Tidskriftsartikel (refereegranskat)abstract
- The complete 1H NMR assignments have been obtained for five mutant proteins of calbindin D9k and the three-dimensional solution structures determined for two of the mutants. The structures have been determined using distance geometry and simulated annealing, with distance constraints from NMR. All mutants have modifications in the first calcium-binding site of calbindin (the N-terminal site designated the pseudo-EF-hand). The 3D structure of the mutant with the most extensive modifications in the pseudo-EF-hand shows that the site has turned inside-out and coordinates calcium as in the normal EF-hand (the C-terminal site). In a pseudo-EF-hand loop the calcium is coordinated by main-chain carbonyls, whereas calcium in the normal EF-hand is coordinated by side-chain carboxylates. The 3D structures and 1H NMR assignments show that in order to accomplish a change in the coordinating ligands of the pseudo-EF-hand the loop must be 12 residues long and have glycine in the sixth position. It does, however, seem possible to have alanine instead of aspartic acid in the first calcium coordinating position. The overall global fold of the proteins has not been affected by the mutations in the calcium-binding site, as compared to the wild-type calbindin D9k [Kördel, J., Skelton, N. J., Akke, M., & Chazin, W. J. (1993) J. Mol. Biol. (in press)]. The structures consist of two helix-calcium-binding loop-helix motifs, the so called EF-hands, and the loops are connected by a short antiparallel β-sheet. All helices are pairwise in an antiparallel orientation.
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| 20. |
- Johansson, Maria U, et al.
(författare)
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Differences in Backbone Dynamics of Two Homologous Bacterial Albumin-binding Modules: Implications for Binding Specificity and Bacterial Adaptation.
- 2002
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 316:5, s. 1083-1099
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Tidskriftsartikel (refereegranskat)abstract
- Proteins G and PAB are bacterial albumin-binding proteins expressed at the surface of group C and G streptococci and Peptostreptococcus magnus, respectively. Repeated albumin-binding domains, known as GA modules, are found in both proteins. The third GA module of protein G from the group G streptococcal strain G148 (G148-GA3) and the second GA module of protein PAB from P.magnus strain ALB8 (ALB8-GA) exhibit 59% sequence identity and both fold to form three-helix bundle structures that are very stable against thermal denaturation. ALB8-GA binds human serum albumin with higher affinity than G148-GA3, but G148-GA3 shows substantially broader albumin-binding specificity than ALB8-GA. The (15)N nuclear magnetic resonance spin relaxation measurements reported here, show that the two GA modules exhibit mobility on the picosecond-nanosecond time scale in directly corresponding regions (loops and termini). Most residues in G148-GA3 were seen to be involved in conformational exchange processes on the microsecond-millisecond time scale, whereas for ALB8-GA such motions were only identified for the beginning of helix 2 and its preceding loop. Furthermore, and more importantly, hydrogen-deuterium exchange and saturation transfer experiments reveal large differences between the two GA modules with respect to motions on the second-hour time scale. The high degree of similarity between the two GA modules with respect to sequence, structure and stability, and the observed differences in dynamics, binding affinity and binding specificity to different albumins, suggest a distinct correlation between dynamics, binding affinity and binding specificity. Finally, it is noteworthy in this context that the module G148-GA3, which has broad albumin-binding specificity, is expressed by group C and G streptococci known to infect all mammalian species, whereas P.magnus with the ALB8-GA module has been isolated only from humans.
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| 21. |
- Johansson, Maria U, et al.
(författare)
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Structure, specificity, and mode of interaction for bacterial albumin-binding modules.
- 2002
-
Ingår i: Journal of Biological Chemistry. - 1083-351X .- 0021-9258. ; 277:10, s. 8114-8120
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Tidskriftsartikel (refereegranskat)abstract
- We have determined the solution structure of an albumin binding domain of protein G, a surface protein of group C and G streptococci. We find that it folds into a left handed three-helix bundle similar to the albumin binding domain of protein PAB from Peptostreptococcus magnus. The two domains share 59% sequence identity, are thermally very stable, and bind to the same site on human serum albumin. The albumin binding site, the first determined for this structural motif known as the GA module, comprises residues spanning the first loop to the beginning of the third helix and includes the most conserved region of GA modules. The two GA modules have different affinities for albumin from different species, and their albumin binding patterns correspond directly to the host specificity of C/G streptococci and P. magnus, respectively. These studies of the evolution, structure, and binding properties of the GA module emphasize the power of bacterial adaptation and underline ecological and medical problems connected with the use of antibiotics.
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| 22. |
- LIZATOVIC, ROBERT, et al.
(författare)
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A de Novo Designed Coiled-Coil Peptide with a Reversible pH-Induced Oligomerization Switch
- 2016
-
Ingår i: Structure. - : Elsevier BV. - 0969-2126. ; 24:6, s. 946-955
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Tidskriftsartikel (refereegranskat)abstract
- Protein conformational switches have many useful applications but are difficult to design rationally. Here we demonstrate how the isoenergetic energy landscape of higher-order coiled coils can enable the formation of an oligomerization switch by insertion of a single destabilizing element into an otherwise stable computationally designed scaffold. We describe a de novo designed peptide that was discovered to switch between a parallel symmetric pentamer at pH 8 and a trimer of antiparallel dimers at pH 6. The transition between pentamer and hexamer is caused by changes in the protonation states of glutamatic acid residues with highly upshifted pKa values in both oligomer forms. The drastic conformational change coupled with the narrow pH range makes the peptide sequence an attractive candidate for introduction of pH sensing into other proteins. The results highlight the remarkable ability of simple-α helices to self-assemble into a vast range of structural states.
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| 23. |
- Lundberg, Peter, et al.
(författare)
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A35Cl--NMR study of the singular anion-binding properties of dromedary hemoglobin
- 1989
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434. ; 999:1, s. 12-8
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Tidskriftsartikel (refereegranskat)abstract
- 35Cl(-)-NMR measurements of chloride binding to carbonmonoxy- and deoxy-dromedary hemoglobin reveal the existence of two classes of chloride-binding sites, one of high and the other of low affinity. Although this situation resembles that described for human hemoglobin, it was found that the number of binding sites as well as the association equilibrium constant for chloride binding are significantly higher in the dromedary protein. This difference may be due to the greater number of basic residues exposed to solvent and to the higher flexibility of dromedary hemoglobin. The two oxygen-linked polyanion-binding sites characteristic of this hemoglobin show competition for some of the high-affinity chloride-binding sites in keeping with their location in the cleft enclosed by the beta chains and between the alpha chains termini. It is suggested that the observed anion-binding properties of dromedary hemoglobin may contribute to the control of the physiological osmotic shock after rehydration.
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| 24. |
- Malmendal, Anders, et al.
(författare)
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Sequence and context dependence of EF-hand loop dynamics. An 15N relaxation study of a calcium-binding site mutant of calbindin D(9k)
- 1998
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 37:8, s. 2586-2595
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Tidskriftsartikel (refereegranskat)abstract
- The influence of amino acid sequence and structural context on the backbone dynamics of EF-hand calcium-binding loops was investigated using 15N spin relaxation measurements on the calcium-free state of the calbindin D(9k) mutant (A14D+A15Δ+P20Δ+N21G+P43M), in which the N-terminal pseudo- EF-hand loop, characteristic of S100 proteins, was engineered so as to conform with the C-terminal consensus EF-hand loop. The results were compared to a previous study of the apo state of the wild-type-like P43G calbindin D(9k) mutant. In the helical regions, the agreement with the P43G data is excellent, indicating that the structure and dynamics of the protein core are unaffected by the substitutions in the N-terminal loop. In the calcium- binding loops, the flexibility is drastically decreased compared to P43G, with the modified N-terminal loop showing a motional restriction comparable to that of the surrounding helixes. As in P43G, the motions in the C-terminal loop are less restricted than in the N-terminal loop. Differences in key hydrogen-bonding interactions correlate well with differences in dynamics and offer insights into the relationship between structure and dynamics of these EF-hand loops. It appears that the entire N-terminal EF-hand is built to form a rigid structure that allows calcium binding with only minor rearrangements and that the structural and dynamical properties of the entire EF-hand- rather than the loop sequence per se-is the major determinant of loop flexibility in this system.
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| 25. |
- Mattinen, M-L, et al.
(författare)
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Quaternary Structure Buildt from Subunuts Combining NMR and Small-Angle X-Ray Scattering Data
- 2002
-
Ingår i: Biophysical Journal. - 1542-0086. ; 83:2, s. 1177-1183
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Tidskriftsartikel (refereegranskat)abstract
- A new principle in constructing molecular complexes from the known high-resolution domain structures joining data from NMR and small-angle x-ray scattering (SAXS) measurements is described. Structure of calmodulin in complex with trifluoperazine was built from N- and C-terminal domains oriented based on residual dipolar couplings measured by NMR in a dilute liquid crystal, and the overall shape of the complex was derived from SAXS data. The residual dipolar coupling data serves to reduce angular degrees of freedom, and the small-angle scattering data serves to confine the translational degrees of freedom. The complex built by this method was found to be consistent with the known crystal structure. The study demonstrates how approximate tertiary structures of modular proteins or quaternary structures composed of subunits can be assembled from high-resolution structures of domains or subunits using mutually complementary NMR and SAXS data.
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| 26. |
- Moparthi, Vamsi, et al.
(författare)
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Functional role of the MrpA- and MrpD-homologous protein subunits in enzyme complexes evolutionary related to respiratory chain complex I.
- 2014
-
Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728. ; 1837:1, s. 178-185
-
Tidskriftsartikel (refereegranskat)abstract
- NADH:quinone oxidoreductase or complex I is a large membrane bound enzyme complex that has evolved from the combination of smaller functional building blocks. Intermediate size enzyme complexes exist in nature that comprise some, but not all of the protein subunits in full size 14-subunit complex I. The membrane spanning complex I subunits NuoL, NuoM and NuoN are homologous to each other and to two proteins from one particular class of Na(+)/H(+) antiporters, denoted MrpA and MrpD. In complex I, these ion transporter protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Using a model system, consisting of Bacillus subtilis MrpA and MrpD deletion strains and a low copy expression plasmid, it was recently demonstrated that NuoN can rescue the strain deleted for MrpD but not that deleted for MrpA, whereas the opposite tendency was seen for NuoL. This demonstrated that the MrpA-type and MrpD-type proteins have unique functional specializations. In this work, the corresponding antiporter-like protein subunits from the smaller enzymes evolutionarily related to complex I were tested in the same model system. The subunits from 11-subunit complex I from Bacillus cereus behaved essentially as those from full size complex I, corroborating that this enzyme should be regarded as a bona fide complex I. The hydrogenase-3 and hydrogenase-4 antiporter-like proteins on the other hand, could substitute equally well for MrpA or MrpD at pH7.4, suggesting that these enzymes have intermediate forms of the antiporter-like proteins, which seemingly lack the functional specificity.
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| 27. |
- Morfeldt, E, et al.
(författare)
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Isolated hypervariable regions derived from streptococcal M proteins specifically bind human C4b-binding protein : implications for antigenic variation.
- 2001
-
Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 167:7, s. 3870-7
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Tidskriftsartikel (refereegranskat)abstract
- Antigenic variation in microbial surface proteins represents an apparent paradox, because the variable region must retain an important function, while exhibiting extensive immunological variability. We studied this problem for a group of streptococcal M proteins in which the approximately 50-residue hypervariable regions (HVRs) show essentially no residue identity but nevertheless bind the same ligand, the human complement regulator C4b-binding protein (C4BP). Synthetic peptides derived from different HVRs were found to retain the ability to bind C4BP, implying that the HVR corresponds to a distinct ligand-binding domain that can be studied in isolated form. This finding allowed direct characterization of the ligand-binding properties of isolated HVRs and permitted comparisons between different HVRs in the absence of conserved parts of the M proteins. Affinity chromatography of human serum on immobilized peptides showed that they bound C4BP with high specificity and inhibition experiments indicated that different peptides bound to the same site in C4BP. Different C4BP-binding peptides did not exhibit any immunological cross-reactivity, but structural analysis suggested that they have similar folds. These data show that the HVR of streptococcal M protein can exhibit extreme variability in sequence and immunological properties while retaining a highly specific ligand-binding function.
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| 28. |
- Nord, Johan, et al.
(författare)
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The C-terminus of dUTPase: observation on flexibility using NMR
- 2001
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Ingår i: FEBS Letters. - 1873-3468. ; 492:3, s. 228-232
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Tidskriftsartikel (refereegranskat)abstract
- The dynamics of the C-terminus of the dUTPases from Escherichia coli and equine infectious anaemia virus (EIAV) were studied by 1H-15N nuclear magnetic resonance spectroscopy. The two enzymes differ with regard to flexibility in the backbone of the 15 most C-terminal amino acid residues, some of which are conserved and essential for enzymic activity. In the bacterial enzyme, the residues closest to the C-terminus are highly flexible and display a correlation time in the nanosecond time range. No similar high flexibility could be detected for the C-terminal part of EIAV dUTPase, indicating a different time range of flexibility.
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| 29. |
- Persson, Jonas, et al.
(författare)
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Subtle sequence differences in a turnour-associated peptide epitope translate into major changes in antigenicity
- 2005
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Ingår i: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; 42:11, s. 1321-1330
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Tidskriftsartikel (refereegranskat)abstract
- Antigenicity, the ability to bind to members of repertoire of diverse immune receptors, is a concept that is poorly characterised with respect to its defining parameters. To learn more about its makeup, we have investigated the ability of two peptides with highly related sequences, derived from the tumour-associated antigen mucin-1, to recruit in vitro members from a large naive repertoire of synthetic human antibody fragments. One of the peptides represents the epitope that is immunodominant in mice. We now demonstrate that the other peptide, which differs from the first only by a very conservative aspartate-threonine to glutamate-serine change, is much less antigenic than the first peptide. This is so despite the fact that there is no observable difference in the tendency of the two peptides to adopt a structure in solution. Furthermore, the peptides differ in their immunodominant parts and the less antigenic peptide selects for antibody fragments targeting residues outside of the epitope considered to be immunodominant in mice. We conclude that subtle sequence changes greatly, affect antigenicity and immunodominance of epitopes in this important tumour-associated antigen. (c) 2005 Elsevier Ltd. All rights reserved.
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| 30. |
- Saveyn, Pieter, et al.
(författare)
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Incomplete lipid chain freezing of sonicated vesicular dispersions of double-tailed ionic surfactants
- 2007
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 23:21, s. 10455-10462
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Tidskriftsartikel (refereegranskat)abstract
- Lipid freezing in dilute sonicated vesicular dispersions was studied using differential scanning calorimetry (DSC) and H-1 NMR. For charged, anionic, or cationic lipids, approximately half of the lipids remain in a fluid state when cooled 20 degrees C below the main chain melting temperature. With a zwitterionic phospholipid, on the other hand, essentially no supercooling of the liquid state was observed. The observations are analyzed in terms of the nucleation and growth of flat solid domains in originally fluid spherical vesicles. As the solid domains grow, the remaining fluid domain is deformed, resulting in a curvature stress. Depending on the vesicle size and the bilayer bending rigidity, the solid domain growth may terminate as the gain in cohesive free energy is balanced by the curvature stress of the remaining fluid domain. It is argued that high bending rigidities are required for, having a significant supercooling, which is why it is only observed for charged lipids.
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| 31. |
- Selander-Sunnerhagen, Maria, et al.
(författare)
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How an epidermal growth factor (EGF)-like domain binds calcium. High resolution NMR structure of the calcium form of the NH2-terminal EGF-like domain in coagulation factor X
- 1992
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 267:27, s. 19642-19649
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Tidskriftsartikel (refereegranskat)abstract
- Domains homologous to the epidermal growth factor (EGF) are important building blocks for extracellular proteins. Proteins containing these domains have been shown to function in such diverse biological processes as blood coagulation, complement activation, and the developmental determination of embryonic cell fates. Many of these proteins require calcium for their biological function. In the case of coagulation factors IX and X and anticoagulants proteins C and S, calcium has been found to bind to the EGF- like domains. We have now determined the three-dimensional structure of the calcium-bound form of the NH2-terminal EGF-like domain in coagulation factor X by two-dimensional NMR and simulated folding. Ligands to the calcium ion are the two backbone carbonyls in Gly-47 and Gly-64, as well as the side chains in Gln-49, erythro-β-hydroxyaspartic acid (Hya) 63, and possibly Asp- 46. The conserved Asp-48 is not a ligand in our present structures. The remaining ligands are assumed to be solvent molecules or, in the intact protein, ligands from neighboring domains. Other proteins interacting in a calcium-dependent manner may also contribute ligands. A comparison with the calcium-free form shows that calcium binding induces strictly local structural changes in the domain. Residues corresponding to the side chain ligands in factor X are conserved in many other proteins, such as the integral membrane protein TAN-1 of human lymphocytes and its developmentally important homolog, Notch, in Drosophila. Calcium binding to EGF-like domains may be crucial for numerous protein-protein interactions involving EGF-like domains in coagulation factors, plasma proteins, and membrane proteins. Therefore, there is reason to believe that this novel calcium site plays an important role in the biochemistry of extracellular proteins.
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| 32. |
- Sorsa, Tia, et al.
(författare)
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Binding of Levosimendan, a Calcium Sensitizer, to Cardiac Troponin C
- 2001
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 276:12, s. 9337-9343
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Tidskriftsartikel (refereegranskat)abstract
- Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca2+-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca2+-loaded regulatory domain of recombinant cTnCC35S was observed. The changes in the NMR spectra of the N-domain of full-length cTnCC35S, due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnCA-Cys, where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnCC35S and cTnCA-Cys. The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca2+-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN 3), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.
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| 33. |
- Sorsa, T, et al.
(författare)
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Interaction of levosimendan with cadiac troponin C in the presence of cardiac troponin I peptides.
- 2003
-
Ingår i: Journal of Molecular and Cellular Cardiology. - 1095-8584. ; 35:9, s. 1055-1061
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between troponin C (TnC) and troponin I (TnI) is essential for the regulation of muscle contraction. There are several binding sites for TnI on TnC that are differentially occupied depending on the phase of the contraction/relaxation cycle. TnI and TnC interact in an antiparallel fashion with each other. The C-domain of cTnC and the N-domain region of cTnI(residues 33–70) always interact under physiological conditions, whereas the interaction between regulatory regions of TnC and TnI (residues 128–166) is calcium dependent. Previously, it has been shown that levosimendan, a calcium sensitizer used as a treatment for acute heart failure, can interact with both domains of isolated cTnC. To understand which interaction is relevant for the mechanism of calcium sensitization, we used a more complete troponin model obtained by complexing cTnI32–79 and cTnI128–180 with calcium-saturated cTnCCS. The cTnI peptides bound to cTnCCS to form a 1:1:1 complex. The interaction of levosimendan with this complex was followed by 1H–15N heteronuclear correlation spectroscopy. It was clear that based on chemical shift changes, cTnI32–79 blocked the levosimendan interaction sites on the C-domain, whereas cTnI128–180 did not compete with levosimendan for the binding site on the N-domain. Hence, the effective binding site of levosimendan on cTnC resulting in the calcium-sensitizing effect is located in the regulatory domain (N-domain).
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| 34. |
- Sorsa, T, et al.
(författare)
-
Stereoselective binding of levosimendan to cardiac troponin C causes Ca2+-sensitization
- 2004
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Ingår i: European Journal of Pharmacology. - : Elsevier BV. - 1879-0712 .- 0014-2999. ; 486:1, s. 1-8
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Tidskriftsartikel (refereegranskat)abstract
- The effects of the Ca2+ sensitizer levosimendan and that of its stereoisomer dextrosimendan on the cardiac contractile apparatus were studied using skinned fibers obtained from guinea pig hearts. Levosimendan was found to be more effective than dextrosimendan in this model. The respective concentrations of levosimendan and dextrosimendan at EC50 were 0.3 and 3 muM. In order to explain the difference in efficacy as Ca2+ sensitizers, the binding of the two stereoisomers on cardiac troponin C was studied by nuclear magnetic resonance in the absence and presence of two peptides of cardiac troponin I. The two stereoisomers interacted with both domains of cardiac troponin C in the absence of cardiac troponin I. In the presence of cardiac troponin I-(32-79) and cardiac troponin I-(128-180), the binding of both levosimendan and dextrosimendan to the C-terminal domain of cardiac troponin C was blocked and only the binding to the N-terminal domain was observable. Differences in the overall binding behavior of the two isomers to cardiac troponin C were highlighted in order to discuss their structure to activity relation. Our data are consistent with the notion that the action of levosimendan as a Ca2+ sensitizer and positive inotrope relates to its stereoselective binding to Ca2+-saturated cardiac troponin C. (C) 2003 Elsevier B.V. All rights reserved.
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| 35. |
- Sperling, Eva, et al.
(författare)
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Functional differentiation of antiporter-like polypeptides in complex I; a site-directed mutagenesis study of residues conserved in MrpA and NuoL but Not in MrpD, NuoM, and NuoN
- 2016
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:7
-
Tidskriftsartikel (refereegranskat)abstract
- It has long been known that the three largest subunits in the membrane domain (NuoL, NuoM and NuoN) of complex I are homologous to each other, as well as to two subunits (MrpA and MrpD) from a Na+ /H+ antiporter, Mrp. MrpA and NuoL are more similar to each other and the same is true for MrpD and NuoN. This suggests a functional differentiation which was proven experimentally in a deletion strain model system, where NuoL could restore the loss of MrpA, but not that of MrpD and vice versa. The simplest explanation for these observations was that the MrpA and MrpD proteins are not antiporters, but rather single subunit ion channels that together form an antiporter. In this work our focus was on a set of amino acid residues in helix VIII, which are only conserved in NuoL and MrpA (but not in any of the other antiporter-like subunits.) and to compare their effect on the function of these two proteins. By combining complementation studies in B. subtilis and 23Na-NMR, response of mutants to high sodium levels were tested. All of the mutants were able to cope with high salt levels; however, all but one mutation (M258I/M225I) showed differences in the efficiency of cell growth and sodium efflux. Our findings showed that, although very similar in sequence, NuoL and MrpA seem to differ on the functional level. Nonetheless the studied mutations gave rise to interesting phenotypes which are of interest in complex I research.
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| 36. |
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| 37. |
- Tossavainen, H, et al.
(författare)
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NMR solution structure of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea
- 2003
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 270:11, s. 2505-2512
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Tidskriftsartikel (refereegranskat)abstract
- The structure of calerythrin, a prokaryotic 20 kDa calcium-binding protein has been determined by solution NMR spectroscopy. Distance, dihedral angle, J coupling, secondary chemical shift, residual dipolar coupling and radius of gyration restraints reveal four EF-hand motifs arranged in a compact globular structure. A tight turn in the middle of the amino acid sequence brings the two halves, each comprising a pair of EF-hands, close together. The structural similarity between calerythrin and the eukaryotic sarcoplasmic calcium-binding proteins is notable.
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| 38. |
- Ullner, Magnus, et al.
(författare)
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Three-Dimensional Structure of the Apo Form of the N-Terminal EGF-like Module of Blood Coagulation Factor X As Determined by NMR Spectroscopy and Simulated Folding
- 1992
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 31:26, s. 5974-5983
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Tidskriftsartikel (refereegranskat)abstract
- The three-dimensional structure of a 42-residue fragment containing the N-terminal EGF-like module of blood coagulation factor X was determined by means of 2D NMR spectroscopy and computer simulation. The spectroscopic data consisted of 370 NOE distances and 27 dihedral angle constraints. These were used to generate peptide conformations by molecular dynamics simulation. The simulations used a novel functional form for the constraint potentials and were performed with two time steps to ensure rapid execution. Apart from preliminary runs to aid assignment of NOEs, 60 runs resulted in 13 accepted structures, which have two antiparallel β sheets, no α helices, and five tight turns. There is no hydrophobic cluster. The root mean square deviation for the backbone of the 13 conformations is 0.65 ± 0.11 Å against their mean conformation. About half of the side chains have well-defined structure. The overall conformation is similar to that of murine EGF.
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