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  • 1. Antuch, W
    et al.
    Berndt, Kurt D
    Eidgenössische Technische Hochschule–Hönggerberg, Zürich, Switzerland.
    Chávez, M A
    Delfín, J
    Wüthrich, K
    The NMR solution structure of a Kunitz-type proteinase inhibitor from the sea anemone Stichodactyla helianthus.1993Ingår i: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 212, s. 675-684Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The solution structure of a 55-amino-acid Kunitz-type proteinase inhibitor, ShPI, purified from the Caribbean sea anemone Stichodactyla helianthus, was determined by NMR spectroscopy. Nearly complete sequence-specific 1H-NMR assignments were obtained at pH 4.6 and 36 degrees C, and stereo-specific assignments were determined for 23 pairs of diastereotopic substituents. A data set of 666 upper distance limit constraints and 122 dihedral angle constraints collected on this basis was used as input for a structure calculation with the program DIANA. Following energy minimization with the program OPAL, the average root-mean-square diviation (RMSD) of the 20 DIANA conformers used to represent the solution structure relative to the mean structure is 61 pm for all backbone atoms N, C alpha and C', and 106 pm for all heavy atoms of residues 2-53. This high-quality solution structure of ShPI has a nearly identical molecular architecture as the bovine pancreatic trypsin inhibitor (BPTI), despite a mere 35% of sequence similarity between the two proteins. Exchange rates measured for 48 out of the 51 backbone amide protons showed that the positions of 20 slowly exchanging amide protons correlate well with hydrogen bonds involving these protons in the energy-minimized solution structure. The solution structure of ShPI is compared to the four homologous proteins for which the three-dimensional structure is also available.

  • 2.
    Berndt, Kurt D
    et al.
    Eidgenössische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
    Beunink, J
    Schröder, W
    Wüthrich, K
    Designed replacement of an internal hydration water molecule in BPTI: structural and functional implications of a glycine-to-serine mutation.1993Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 32, s. 4564-4570Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The three-dimensional structure of the basic pancreatic trypsin inhibitor (BPTI) contains four internal water molecules, which form a total of nine intermolecular hydrogen bonds with the BPTI polypeptide chain. To investigate the effect of such internal hydration on protein structure and stability, we displaced one of the internal water molecules in a recombinant BPTI analogue, BPTI(G36S), in which Gly 36 is replaced by serine. The replacement of a water molecule by the seryl side chain was established by the absence of the protein-water nuclear Overhauser effects (NOE) that had been attributed to the water molecule near Gly 36 in wild-type BPTI and by the presence of new, intramolecular NOEs to the hydroxyl proton of Ser 36. BPTI(G36S) has slightly reduced thermal stability compared to BPTI, corresponding to a destabilization by delta (delta G) approximately 0.7 kcal/M in 6 M guanidinium hydrochloride solution. Additionally, the stabilities of the complexes formed between BPTI(G36S) and trypsin, plasmin, or kallikrein are significantly reduced when compared to the corresponding complexes with wild-type BPTI.

  • 3.
    Berndt, Kurt D
    et al.
    Eidgenossische TH-Honggerberg, Zürich, Switzerland.
    Güntert, P
    Wüthrich, K
    Nuclear-Magnetic-Resonance Solution Structure of Dendrotoxin-K from the Venom of Dendroaspis-Polylepis-Polylepis1993Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 234, nr 3, s. 735-750Artikel i tidskrift (Refereegranskat)
  • 4.
    Berndt, Kurt D
    et al.
    Eidgenösische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
    Güntert, Peter
    Orbons, Leonard P.M.
    Wüthrich, Kurt
    Determination of a high-quality nuclear magnetic resonance solution structure of the bovine pancreatic trypsin inhibitor and comparison with three crystal structures1992Ingår i: Journal of Molecular Biology, Vol. 227, s. 757-775Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A high-quality three-dimensional structure of the bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution was determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and compared to the three available high-resolution X-ray crystal structures. A newly collected input of 642 distance constraints derived from nuclear Overhauser effects and 115 dihedral angle constraints was used for the structure calculations with the program DIANA, followed by restrained energy minimization with the program AMBER. The BPTI solution structure is represented by a group of 20 conformers with an average root-mean-square deviation (RMSD) relative to the mean solution structure of 0.43 A for backbone atoms and 0.92 A for all heavy atoms of residues 2 to 56. The pairwise RMSD values of the three crystal structures relative to the mean solution structure are 0.76 to 0.85 A for the backbone atoms and 1.24 to 1.33 A for all heavy atoms of residues 2 to 56. Small local differences in backbone atom positions between the solution structure and the X-ray structures near residues 9, 25 to 27, 46 to 48 and 52 to 58, and conformational differences for individual amino acid side-chains were analyzed for possible correlations with intermolecular protein-protein contacts in the crystal lattices, using the pairwise RMSD values among the three crystal structures as a reference.

  • 5.
    Berndt, Kurt D
    et al.
    Eidgenössische TH-Hönggerberg, Zürich, Switzerland.
    Güntert, Peter
    Wüthrich, Kurt
    Conformational sampling by NMR solution structures calculated with the program DIANA evaluated by comparison with long-time molecular dynamics calculations in explicit water1996Ingår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 24, s. 304-313Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The NMR solution structure of bovine pancreatic trypsin inhibitor (BPTI) obtained by distance geometry calculations with the program DIANA is compared with groups of conformers generated by molecular dynamics (MD) simulations in explicit water at ambient temperature and pressure. The MD simulations started from a single conformer and were free or restrained either by the experimental NOE distance restraints or by time-averaged restraints; the groups of conformers were collected either in 10 ps intervals during 200 ps periods of simulation, or in 50 ps intervals during a 1 ns period of simulation. Overall, these comparisons show that the standard protein structure determination protocol with the program DIANA provides a picture of the protein structure that is in agreement with MD simulations using "realistic" potential functions over a nanosecond timescale. For well-constrained molecular regions there is a trend in the free MD simulation of duration 1 ns that the sampling of the conformation space is slightly increased relative to the DIANA calculations. In contrast, for surface-exposed side-chains that are less extensively constrained by the NMR data, the DIANA conformers tend to sample larger regions of conformational space than conformers selected from any of the MD trajectories. Additional insights into the behavior of surface side-chains come from comparison of the MD runs of 200 ps or 1 ns duration. In this time range the sampling of conformation space by the protein surface depends strongly on the length of the simulation, which indicates that significant side-chain transitions occur on the nanosecond timescale and that much longer simulations will be needed to obtain statistically significant data on side-chain dynamics.

  • 6. Brunne, R M
    et al.
    Berndt, Kurt D
    ETH Hönggerberg, Zürich, Switzerland.
    Güntert, P
    Wüthrich, K
    van Gunsteren, W F
    Güntert, P
    Wüthrich, K
    Van Gunsteren, W F
    Structure and internal dynamics of the bovine pancreatic trypsin inhibitor in aqueous solution from long-time molecular dynamics simulations1995Ingår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 23, nr 1, s. 49-62Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Structural and dynamic properties of bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution are investigated using two molecular dynamics (MD) simulations: one of 1.4 ns length and one of 0.8 ns length in which atom-atom distance bounds derived from NMR spectroscopy are included in the potential energy function to make the trajectory satisfy these experimental data more closely. The simulated properties of BPTI are compared with crystal and solution structures of BPTI, and found to be in agreement with the available experimental data. The best agreement with experiment was obtained when atom-atom distance restraints were applied in a time-averaged manner in the simulation. The polypeptide segments found to be most flexible in the MD simulations coincide closely with those showing differences between the crystal and solution structures of BPTI.

  • 7. Chin, T M
    et al.
    Berndt, Kurt D
    Universtity of Chicago, USA.
    Yang, N C C
    Self-Assembling Hexameric Helical Bundle Forming Peptides1992Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 114, nr 6, s. 2279-2280Artikel i tidskrift (Refereegranskat)
  • 8. Güntert, P
    et al.
    Berndt, Kurt D
    Eidgenössische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
    Wüthrich, K
    The Program Asno for Computer-Supported Collection of Noe Upper Distance Constraints as Input for Protein-Structure Determination1993Ingår i: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 3, nr 5, s. 601-606Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A new program, ASNO ('ASsign NOes'), for computer-supported NOE cross-peak assignments is described. ASNO is used for structure refinement in several rounds of NOESY cross-peak assignments and 3D structure calculations, where the preliminary structures are used as a reference to resolve ambiguities in NOE assignments which are otherwise based on the chemical shifts available from the sequence-specific resonance assignments. The practical use of ASNO for proteins is illustrated with the structure determination of Dendrotoxin K from Dendroaspis polylepis polylepis.

  • 9. Hammarström, A
    et al.
    Berndt, Kurt D
    Karolinska Institutet.
    Sillard, R
    Adermann, K
    Otting, G
    Solution structure of a naturally-occurring zinc-peptide complex demonstrates that the N-terminal zinc-binding module of the Lasp-1 LIM domain is an independent folding unit1996Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 35, nr 39, s. 12723-12732Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The three-dimensional solution structure of the 1:1 complex between the synthetic peptide ZF-1 and zinc was determined by H-1 NMR spectroscopy. The peptide, initially isolated from pig intestines, is identical in sequence to the 30 N-terminal amino acid residues of the human protein Lasp-1 belonging to the LIM domain protein family. The final set of 20 energy-refined NMR conformers has an average rmsd relative to the mean structure of 0.55 Angstrom for the backbone atoms of residues 3-30, Calculations without zinc atom constraints unambiguously identified Cys 5, Cys 8, His 26, and Cys 29 as the zinc-coordinating residues. LIM domains consist of two sequential zinc-binding modules and the NMR structure of the ZF-1(-)zinc complex is the first example of a structure of an isolated module. Comparison with the known structures of the N-terminal zinc-binding modules of both the second LIM domain of chicken CRP and rat GRIP with which ZF-1 shares 50% and 43% sequence identity, respectively, supports the notion that the zinc-binding modules of the LIM domain have a conserved structural motif and identifies local regions of structural diversity. The similarities include conserved zinc-coordinating residues, a rubredoxin knuckle involving Cys 5 and Cys 8, and the coordination of the zinc ion by histidine N-delta in contrast to the more usual coordination by N-epsilon observed for other zinc-finger domains, The present structure determination of the ZF-1(-)zinc complex establishes the N-terminal half of a LIM domain as an independent folding unit. The structural similarities of N- and C-terminal zinc-binding modules of the LIM domains, despite limited sequence identity, lead to the proposal of a single zinc-binding motif in LIM domains. The coordinates are available from the Brookhaven protein data bank, entry 1ZFO.

  • 10. Johansson, J
    et al.
    Gudmundsson, G H
    Rottenberg, M E
    Berndt, Kurt D
    Karolinska Institutet.
    Agerberth, B
    Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-371998Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 273, nr 6, s. 3718-3724Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The influence of ion composition, pH, and peptide concentration on the conformation and activity of the 37-residue human antibacterial peptide LL-37 has been studied. At micromolar concentration in water, LL-37 exhibits a circular dichroism spectrum consistent with a disordered structure. The addition of 15 mM HCO3-, SO42-, or CF3CO2- causes the peptide to adopt a helical structure, with approximately equal efficiency, while 160 mM Cl- is less efficient, A cooperative transition from disordered to helical structure is observed as the peptide concentration is increased, consistent with formation of an oligomer, The extent of alpha-helicity correlates with the antibacterial activity of LL-37 against both Gram-positive and Gram-negative bacteria. Two homologous peptides, FF-33 and SK-29, containing 4 and 8 residue deletions at the N terminus, respectively, require higher concentrations of anions for helix formation and are less active than LL 37 against Escherichia coli D21. Below pH 5, the helical content of LL-37 gradually decreases, and at pH 2 it is entirely disordered, In contrast, the helical structure is retained at pH over 13. The minimal inhibitory concentration of LL-37 against E. coli is 5 mu M, and at 13-25 mu M the peptide is cytotoxic against several eukaryotic cells, In solutions containing the ion compositions of plasma, intracellular fluid, or interstitial fluid, LL-37 is helical, and hence it could pose a danger to human cells upon release. However, in the presence of human serum, the antibacterial and the cytotoxic activities of LL-37 are inhibited.

  • 11. Kaiser, L
    et al.
    Velickovic, T C
    Badia-Martinez, D
    Adedoyin, J
    Thunberg, S
    Hallen, D
    Berndt, Kurt D
    Karolinska Institutet.
    Gronlund, H
    Gafvelin, G
    van Hage, M
    Achour, A
    Structural characterization of the tetrameric form of the major cat allergen Fel d 12007Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 370, nr 4, s. 714-727Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Felis domesticus allergen 1(Fel d 1) is a 35 kDa tetrameric glycoprotein formed by two heterodimers which elicits IgE responses in 95% of patients with allergy to cat. We have previously established in vitro conditions for the appropriate folding of recombinant Fel d 1 using a direct linkage of chain 1 to chain 2 (construct Fel d 1 (1+2)) and chain 2 to chain 1 (construct Fel d 1 (2+1)). Although the crystal structure of Fel d 1 (2+1) revealed a striking structural similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties, no functional tetrameric form of Fel d 1 could be identified. Here we present the crystal structure of the Fel d 1 (1+2) tetramer at 1.6 A resolution. Interestingly, the crystal structure of tetrameric Fel d 1 reveals two different calcium-binding sites. Symmetrically positioned on each side of the Fel d 1 tetramer, the external Ca(2+)-binding sites correspond to a putative Ca(2+)-binding site previously suggested for uteroglobin. The second Ca(2+)-binding site lies within the dimerization interface, stabilizing the formation of the Fel d 1 tetramer, and inducing important local conformational changes that directly govern the shape of two water-filled cavities. The crystal structure suggests a potential portal for an unknown ligand. Alternatively, the two cavities could be used by the allergen as a conditional inner space allowing for the spatial rearrangement of centrally localized side-chains, such as Asp130, without altering the overall fold of the molecule. The striking structural similarity of the major cat allergen to uteroglobin, coupled to the identification in the present study of a common Ca(2+)-binding site, let us speculate that Fel d 1 could provoke an allergic response through the modulation of phospholipase A2, by sequestering Ca ions in a similar manner as previously suggested for uteroglobin.

  • 12. Knapp, S
    et al.
    Karshikoff, A
    Berndt, Kurt D
    Karolinska Institutet.
    Christova, P
    Atanasov, B
    Ladenstein, R
    Thermal unfolding of the DNA-binding protein Sso7d from the hyperthermophile Sulfolobus solfataricus1996Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 264, nr 5, s. 1132-1144Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Thermal unfolding of the small hyperthermophilic DNA-binding protein Sso7d was studied by circular dichroism spectroscopy and differential scanning calorimetry. The unfolding transition can be described by a reversible two state process. Maximum stability was observed in the region between pH 4.5 and 7.0 where Sso7d unfolds with a melting temperature between 370.8 to 371.9 K and an unfolding enthalpy between 62.9 and 65.4 kcal/mol. The heat capacity differences between the native and the heat denatured states obtained by differential scanning calorimetry (620 cal/(mol K)) and circular dichroism spectroscopy (580 cal/(mol K)) resulted in comparable values. The thermodynamic reason for the high melting temperature of Sso7d is the shallow stability curve with a broad free energy maximum, corresponding to the relatively small heat capacity change which was obtained. The calculated stability curve shows that Sso7d has, despite of its high melting temperature, an only moderate intrinsic stability, which reaches its maximum (approximate to 7 kcal/mol) at 282 K. Sso7d is particularly poorly stabilized (approximate to 1 kcal/mol) at the maximum physiological growth temperature of Sulfolobus solfataricus. Sso7d has furthermore untypically low specific enthalpy (0.99 kcal/(mol residue)) and entropy (2.99 cal/(mol K)) values at convergence temperatures. No significant differences in thermal stability of the partially methylated Sso7d from Sulfolobus solfataricus and the cloned non-methylated form of the protein expressed in Escherichia coli were observed. (C) 1996 Academic Press Limited

  • 13. Knapp, S
    et al.
    Mattson, P T
    Christova, P
    Berndt, Kurt D
    Karolinska Institutet.
    Karshikoff, A
    Vihinen, M
    Smith, C I E
    Ladenstein, R
    Thermal unfolding of small proteins with SH3 domain folding pattern1998Ingår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 31, nr 3, s. 309-319Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy, The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec), At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk-, Itk-, and Tec-SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12-16 kJ/mol but show quite high melting temperatures. Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities. (C) 1998 Wiley-Liss, Inc.

  • 14. Liepinsh, E
    et al.
    Berndt, Kurt D
    Karolinska Institutet.
    Sillard, R
    Mutt, V
    Otting, G
    Solution Structure and Dynamics of Pec-60, a Protein of the Kazal Type Inhibitor Family, Determined by Nuclear-Magnetic-Resonance Spectroscopy1994Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 239, nr 1, s. 137-153Artikel i tidskrift (Refereegranskat)
  • 15. Nilsson, Ola B.
    et al.
    Adedoyin, Justus
    Rhyner, Claudio
    Neimert-Andersson, Theresa
    Grundström, Jeanette
    Berndt, Kurt D
    Karolinska Institutet.
    Crameri, Reto
    Grönlund, Hans
    In vitro evolution of allergy vaccine candidates, with maintained structure, but reduced B cell and T cell activation capacity2011Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, artikel-id e24558Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients.

  • 16. Nordstrand, K
    et al.
    Åslund, F
    Holmgren, A
    Otting, G
    Berndt, Kurt D
    Karolinska Institutet.
    NMR structure of Escherichia coli glutaredoxin 3-glutathione mixed disulfide complex: Implications for the enzymatic mechanism1999Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 286, nr 2, s. 541-552Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Glutaredoxins (Grxs) catalyze reversible oxidation/reduction of protein disulfide groups and glutathione-containing mixed disulfide groups via an active site Grx-glutathione mixed disulfide (Grx-SG) intermediate. The NMR solution structure of the Escherichia coli Grx3 mixed disulfide with glutathione (Grx3-SG) was determined using a C14S mutant which traps this intermediate in the redox reaction. The structure contains a thioredoxin fold, with a well-defined binding site for glutathione which involves two intermolecular backbone-backbone hydrogen bonds forming an antiparallel intermolecular beta-bridge between the protein and glutathione. The solution structure of E. coli Grx3-SG also suggests a binding site for a second glutathione in the reduction of the Grx3-SG intermediate, which is consistent with the specificity of reduction observed in Grxs. Molecular details of the structure in relation to the stability of the intermediate and the activity of Grx3 as a reductant of glutathione mixed disulfide groups are discussed. A comparison of glutathione binding in Grx3-SG and ligand binding in other members of the thioredoxin superfamily is presented, which illustrates the highly conserved intermolecular interactions in this protein family. (C) 1999 Academic Press.

  • 17. Nordstrand, K
    et al.
    Åslund, F
    Meunier, S
    Holmgren, A
    Otting, G
    Berndt, Kurt D
    Karolinska Institutet.
    Direct NMR observation of the Cys-14 thiol proton of reduced Escherichia coli glutaredoxin-3 supports the presence of an active site thiol-thiolate hydrogen bond1999Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 449, nr 2-3, s. 196-200Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The active site of Escherichia coli glutaredoxin-3 (Grx3) consists of two redox active cysteine residues in the sequence -C-11-P-Y-C-14-H-. The H-1 NMR resonance of the cysteine thiol proton of Cys-14 in reduced Grx3 is observed at 7.6 ppm. The large downfield shift and NOEs observed with this thiol proton resonance suggest the presence of a hydrogen bond with the Cvs-ll thiolate, which is shown to have an abnormally low pK(a) value. A hydrogen bond would also agree,vith activity data of Grx3 active site mutants. Furthermore, the activity is reduced in a Grx3 H15V mutant, indicating electrostatic contributions to the stabilization of the Cys-ll thiolate, (C) 1999 Federation of European Biochemical Societies.

  • 18. Wade, D
    et al.
    Palma, M
    Flock, J I
    Berndt, Kurt D
    Karolinska Institutet.
    Silberring, J
    Galaktionov, S G
    Structural analysis of Efb, a fibrinogen binding protein of Staphylococcus aureus1998Ingår i: Protein peptide letters, ISSN 0929-8665, E-ISSN 1875-5305, Vol. 5, nr 4, s. 199-206Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Staphylococcus aureus produces and secretes a small, basic protein, designated Efb, that binds to fibrinogen and seems to be required for virulence of the organism. A 3D model of Efb was developed, and it predicts that the C-terminal half of the protein contains substantial alpha-helical content. CD analysis of Efb yielded a value of 40-41% alpha-helix. Calcium and zinc both influence the interactions between Efb and fibrinogen, and an analysis of the amino acid sequence of Efb revealed the presence of consensus sequences for the binding of both metals.

  • 19. Wüthrich, Kurt
    et al.
    Güntert, Peter
    Berndt, Kurt D.
    Computer-supported Structure Determination of Proteins in Solution Illustrated with Studies of Protein Proteinase Inhibitors1993Ingår i: Innovations in Proteinases and Their Inhibitors / [ed] Avilés, Francese X., Berlin: Walter de Gruyter, 1993, s. 407-Kapitel i bok, del av antologi (Övrigt vetenskapligt)
  • 20. Åslund, F.
    et al.
    Berndt, Kurt D
    Karolinska Institutet.
    Holmgren, A.
    Redox potentials of glutaredoxins and other thiol-disulfide oxidoreductases of the thioredoxin superfamily determined by direct protein-protein redox equilibria1997Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, nr 49, s. 30780-30786Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Glutaredoxins belong to the thioredoxin superfamily of structurally similar thiol-disulfide oxidoreductases catalyzing thiol-disulfide exchange reactions via reversible oxidation of two active-site cysteine residues separated by two amino acids (CX1X2C). Standard state redox potential (E degrees ') values for glutaredoxins are presently unknown, and use of glutathione/glutathione disulfide (GSH/GSSG) redox buffers for determining E degrees ' resulted in variable levels of GSH-mixed disulfides. To overcome this complication, we have used reverse-phase high performance liquid chromatography to separate and quantify the oxidized and reduced forms present in the thiol-disulfide exchange reaction at equilibrium after mixing one oxidized and one reduced protein. This allowed for direct and quantitative pair-wise comparisons of the reducing capacities of the proteins and mutant forms. Equilibrium constants from pair-wise reaction with thioredoxin or its P34H mutant, which have accurately determined E degrees ' values from their redox equilibrium with NADPH catalyzed by thioredoxin reductase, allowed for transformation into standard state values. Using this new procedure, the standard state redox potentials for the Escherichia coli glutaredoxins 1 and 3, which contain identical active site sequences CPYC, were found to be E degrees ' = -233 and -198 mV, respectively. These values were confirmed independently by using the thermodynamic linkage between the stability of the disulfide bond and the stability of the protein to denaturation. Comparison of calculated E degrees ' values from a number of proteins ranging from -270 mV for E. coli Trx to -124 mV for DsbA obtained using this method with those determined using glutathione redox buffers provides independent confirmation of the standard state redox potential of glutathione as -240 mV. Determining redox potentials through direct protein-protein equilibria is of general interest as it overcomes errors in determining redox potentials calculated from large equilibrium constants with the strongly reducing NADPH or by accumulating mixed disulfides with GSH.

  • 21. Åslund, F
    et al.
    Nordstrand, K
    Berndt, Kurt D
    Karolinska Institutet.
    Nikkola, M
    Bergman, T
    Ponstingl, H
    Jornvall, H
    Otting, G
    Holmgren, A
    Glutaredoxin-3 from Escherichia coli: Amino acid sequence, H-1 and N-15 NMR assignments, and structural analysis1996Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 271, nr 12, s. 6736-6745Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The primary and secondary structure of glutaredoxin-3 (Grx3), a glutathione-disulfide oxidoreductase from Escherichia coli, has been determined. The amino acid sequence of Grx3 consists of 82 residues and contains a redox-active motif, Cys-Pro-Tyr-Cys, typical of the glutaredoxin family. Sequence comparison reveals a homology (33% identity) to that of glutaredoxin-1 (Grx1) from E. coli as well as to other members of the thioredoxin superfamily. in addition to the active site cysteine residues, Grx3 contains one additional cysteine (Cys(65)) corresponding to one of the two non-active site (or structural) cysteine residues present in mammalian glutaredoxins. The sequence-specific H-1 and N-15 nuclear magnetic resonance assignments of reduced Grx3 have been obtained. From a combined analysis of chemical shifts, (3)J(HN alpha) coupling constants, sequential and medium range NOEs, and amide proton exchange rates, the secondary structure of reduced Grx3 was determined and found to be very similar to that inferred from amino acid sequence comparison to homologous proteins. The consequences of the proposed structural similarity to Grx1 are that Grx3, while possessing a largely intact GSH binding cleft, would have a very different spatial distribution of charged residues, most notably surrounding the active site cysteine residues and occurring in the proposed hydrophobic protein-protein interaction area. These differences may contribute to the observed very low K-cat of Grx3 as a reductant of insulin disulfides or as a hydrogen donor for ribonucleotide reductase. Thus, despite an identical active site disulfide motif and a similar secondary structure and tertiary fold, Grx3 and Grxl display large functional differences in in vitro protein disulfide oxide-reduction reactions.

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