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T Drakenberg

Publications and source records attributed to T Drakenberg.

At least 73 records · Page 4Linked to original sources
PubMed1993

Two-dimensional nuclear magnetic resonance study of the beta-casein peptide 1-25: resonance assignments and secondary structure.

The N-terminal 25 amino-acid residue peptide (beta-CN 1-25) obtained from a tryptic digest of bovine beta-casein was studied using two-dimensional NMR techniques. Complete sequence-specific assignment of the 1H-NMR spectrum was performed both in the presence and absence of 22 mM Ca2+. The NMR data supported earlier findings that this segment of beta-casein is highly flexible and adopts multiple conformations. The observed pattern of sequential NOEs indicated that the peptide did not contain stable folded structures. However, the structure was neither that of a fully-extended peptide nor a so-called random coil. The region Ile-12 to SerP-15 showed an enhanced population of extended structure. Furthermore, addition of Ca2+ ions induced chemical-shift changes and apparently decreased the population of conformations with extended structure in the region SerP-18 to Ile-23.

Amino Acid Sequence↗
PubMed1993

The solution structures of mutant calbindin D9k's, as determined by NMR, show that the calcium-binding site can adopt different folds.

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 beta-sheet. All helices are pairwise in an antiparallel orientation.

Amino Acid Sequence↗
PubMed1993

The cis/trans interconversion of the calcium regulating hormone calcitonin is catalyzed by cyclophilin.

The cytosolic peptidyl-prolyl cis/trans isomerase cyclophilin from pig kidney can accelerate catalytically the cis/trans isomerization of prolyl peptide bonds. One- and two-dimensional 1H NMR spectroscopy was used to prove that the polypeptide hormone calcitonin is a substrate for cyclophilin. Isomerization of only one of the two prolyl peptide bonds is catalyzed significantly. The efficiency of catalysis was calculated by lineshape analysis and NOESY spectroscopy. Cyclosporin A completely blocks the effect of the enzyme on the conformational dynamics of the polypeptide.

Amino Acid Isomerases↗
PubMed1993

Proton nuclear magnetic resonance sequential assignments and secondary structure of an immunoglobulin light chain-binding domain of protein L.

The 1H NMR assignments have been made for the immunoglobulin (Ig) light chain-binding B1 domain of protein L from Peptostreptococcus magnus. The secondary structure elements and the global folding pattern were determined from nuclear Overhauser effects, backbone coupling constants, and slowly exchanging amide protons. The B1 domain was found to be folded into a globular unit of 61 amino acid residues, preceded by a 15 amino acid long disordered N-terminus. The folded portion of the molecule contains a four-stranded beta-sheet spanned by a central alpha-helix. The fold is similar to the IgG-binding domains of streptococcal protein G, despite the fact that the binding sites on immunoglobulins for the two proteins are different; protein G binds IgG through the constant (Fc) part of the heavy chain, whereas protein L has affinity for the variable domain of Ig light chains.

Amino Acid Sequence↗
PubMed1992

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.

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-beta-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.

Amino Acid Sequence↗
PubMed1992

Convergent evolution among immunoglobulin G-binding bacterial proteins.

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.

Amino Acid Sequence↗
PubMed1992

Calcium-43 NMR studies of calcium-binding lysozymes and alpha-lactalbumins.

The calcium-binding properties of equine and pigeon lysozyme as well as those of bovine and human alpha-lactalbumin were investigated by 43Ca NMR spectroscopy. All proteins were found to contain one high-affinity calcium-binding site. The chemical shifts, line widths, relaxation times (T1 and T2), and quadrupole coupling constants for the respective 43Ca NMR signals were quite similar; this is indicative of a high degree of homology between the strong calcium-binding sites of these four proteins. The measured chemical shifts (delta approximately -3 to -7 ppm) and quadrupole coupling constants (chi approximately 0.7-0.8 MHz) are quite distinct from those observed for typical EF-hand calcium-binding proteins, suggesting a different geometry for the calcium-binding loops. The correlation times for bound calcium ions in these proteins were on the order of 4-8 ns, indicating that the flexibilities of these binding sites are limited. The apparent pKa values for the high-affinity sites ranged from 3.4 to 4.7, confirming the participation of carboxylate-containing residues in the coordination of the calcium ion. Competition experiments with EDTA showed that the affinities of these proteins for calcium follow the series bovine alpha-lactalbumin approximately human alpha-lactalbumin greater than pigeon lysozyme greater than equine lysozyme (KD approximately 5 x 10(-8) to 10(-6) M). Evidence for the existence of a second weak calcium-binding site (KD = 3 x 10(-3) M) was obtained for bovine alpha-lactalbumin, but not for the other proteins studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

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.

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 beta sheets, no alpha 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 A against their mean conformation. About half of the side chains have well-defined structure. The overall conformation is similar to that of murine EGF.

Amino Acid Sequence↗
PubMed1992

Prokaryotic calcium-binding protein of the calmodulin superfamily. Calcium binding to a Saccharopolyspora erythraea 20 kDa protein.

The EF-hand calcium-binding protein from Saccharopolyspora erythraea has been shown, using 113Cd NMR, to possess three Cd(2+)-ion binding sites. This indicates that of the four EF-hand motifs in the molecule, one (probably site 2) is unable to bind Cd(2+)-ions. Data from the titration of the protein with Ca2+, in the presence of Quin2, were fitted to a curve calculated on the assumption that the protein contains three high affinity Ca2+ binding sites, two of which (pK1 = 8.0, pK2 = 9.0) are strongly cooperative, and one single site (pK3 = 7.5). Preliminary 1H NMR experiments indicate marked structural changes upon Ca(2+)-binding.

Binding Sites↗
PubMed1992

Three-dimensional solution structure of Ca(2+)-loaded porcine calbindin D9k determined by nuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of native, intact porcine calbindin D9k has been determined by distance geometry and restrained molecular dynamics calculations using distance and dihedral angle constraints obtained from 1H NMR spectroscopy. The protein has a well-defined global fold consisting of four helices oriented in a pairwise antiparallel manner such that two pairs of helix-loop-helix motifs (EF-hands) are joined by a linker segment. The two EF-hands are further coupled through a short beta-type interaction between the two Ca(2+)-binding loops. Overall, the structure is very similar to that of the highly homologous native, minor A form of bovine calbindin D9k determined by X-ray crystallography [Szebenyi, D. M. E., & Moffat, K. (1986) J. Biol. Chem. 261, 8761-8776]. A model structure built from the bovine calbindin D9k crystal structure shows several deviations larger than 2 A from the experimental distance constraints for the porcine protein. These structural differences are efficiently removed by subjecting the model structure to the experimental distance and dihedral angle constraints in a restrained molecular dynamics protocol, thereby generating a model that is very similar to the refined distance geometry derived structures. The N-terminal residues of the intact protein that are absent in the minor A form appear to be highly flexible and do not influence the structure of other regions of the protein. This result is important because it validates the conclusions drawn from the wide range of studies that have been carried out on minor A forms rather than the intact calbindin D9k.

Amino Acid Sequence↗
PubMed1992

Ca2+ binding environments on natural and synthetic polymeric DNA's.

Here are reported 43Ca nmr chemical shift and line width measurements obtained during 43CaClO4 titrations of two natural and two synthetic polymeric DNA's. Titrations of the natural DNA's demonstrate the existence of at least two classes of bound 43Ca2+. The 43Ca2+ nmr relaxation and chemical shift behavior observed during titration of C. perfringens DNA (31%GC) is dominated by a delocalized, non-specific interaction. In contrast, titration of M. lysodeikticus DNA (72% GC) indicates that a small fraction of the 43Ca2+ experiences significant motional retardation and/or an increase in the electric field gradient when associated to the DNA, and thus appears to be locally bound to discrete sites on the DNA. These results, and previous results for calf thymus DNA (39% GC) demonstrate that higher GC content correlates with an increase in favorable Ca2+ binding environments. Titrations of synthetic DNA demonstrate that Ca2+ binding is remarkably sensitive to local DNA structure.

Animals↗
PubMed1991

Mutation of the pseudo-EF-hand of calbindin D9k into a normal EF-hand. Biophysical studies.

The two Ca(2+)-binding sites in calbindin D9k, a protein belonging to the calmodulin superfamily of intracellular proteins, have slightly different structure. The C-terminal site (amino acids 54-65) is a normal EF-hand as in the other proteins of the calmodulin superfamily, while the N-terminal site (amino acids 14-27) contains two additional amino acids, one of which is a proline. We have constructed and studied five mutants of calbindin D9k modified in the N-terminal site. In normal EF-hand structures the first amino acid to coordinate calcium is invariantly an Asp. For this reason Ala15, is exchanged by an Asp in all mutants and the mutants also contain various other changes in this site. The mutants have been characterized by 43Ca, 113Cd and 1H NMR and by the determination of the calcium binding constants using absorption chelators. In two of the mutants (one where Ala14 is deleted, Ala15 is replaced by Asp and Pro20 is replaced by Gly, the other where, in addition, Asn21 is deleted), we find that the structure has changed considerably compared to the wild-type calbindin. The NMR results indicate that the calcium coordination has changed to mainly side-chain carboxyls, from being octahedrally coordinated by mainly back-bone carbonyls, and/or that the coordination number has decreased. The N-terminal site has thus been turned into a normal EF-hand, in which the calcium ion is coordinated by side-chain carboxyls. Furthermore, the calcium binding constants of these two mutant proteins are almost as high as in the wild-type calbindin D9k. That is, the extensive alterations in the N-terminal site have not disrupted the calcium binding ability of the proteins.

Amino Acid Sequence↗
PubMed1991

Peptidyl-prolyl cis-trans isomerase activity as studied by dynamic proton NMR spectroscopy.

Recently the identity of the peptidyl-prolyl cis-trans isomerase (PPIase), which accelerates the cis/trans isomerization of prolyl peptide bonds and cyclophilin, the binding protein for the immunosuppressive drug Cyclosporin A (CsA), was discovered. The PPIase catalysis toward the substrate Suc-Ala-Phe-Pro-Phe-pNA has been studied by 1H NMR spectroscopy. Using the bandshape analysis technique the rate of interconversion between the cis and trans isomers of the substrate could be measured in the presence of PPIase and under equilibrium conditions. The acceleration is inhibited by equimolar amounts of CsA. The results provide evidence that the PPIase catalysis is more complex than a simple exchange between two states.

Amino Acid Isomerases↗
PubMed1991

Electrostatic contributions to the binding of Ca2+ in calbindin D9k.

A set of accurate experimental data is provided for Ca2+ ion binding to calbindin D9k, a protein in the calmodulin superfamily of intracellular regulatory proteins. The study comprises both the role of protein surface charges and the effects of added electrolyte. The two macroscopic Ca2(+)-binding constants K1 and K2 are determined for the wild-type and eight mutant calbindins in 0, 0.05, 0.10, and 0.15 M KCl from titrations in the presence of Quin 2 or 5,5'-Br2BAPTA. The mutations involve replacement of surface carboxylates (of Glu17, Asp19, Glu26, and Glu60) with the corresponding amides. It is found that K1K2 may decrease by a factor of up to 2.5 x 10(5) (triple mutant in 0.15 M KCl as compared to the wild-type protein in 0 M KCl). Ca2(+)-binding constants of the individual Ca2+ sites (microscopic binding constants) have also been determined. The positive cooperativity of Ca2+ binding, previously observed at low salt concentration [Linse et al. (1987) Biochemistry 26, 6723-6735], is also present at physiological ionic strength and amounts to 5 kJ.mol-1 at 0.15 M KCl. The electrolyte concentration and some of the mutations are found to affect the cooperativity. 39K NMR studies show that K+ binds weakly to calbindin. Two-dimensional 1H NMR studies show, however, that potassium binding does not change the protein conformation, and the large effect of KCl on the Ca2+ affinity is thus of unspecific nature. Two-dimensional 1H NMR has also been used to assess the structural consequences of the mutations through assignments of the backbone NH and C alpha H resonances of six mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Ca2+ binding in proteins of the calmodulin superfamily: cooperativity, electrostatic contributions and molecular mechanisms.

In a large number of intracellular regulatory proteins of the calmodulin superfamily a pair of closely interacting helix-loop-helix Ca2+ binding sites ('EF hands') constitute the functional unit--an arrangement that enables cooperative binding. We have recently made detailed experimental studies of the binding of Ca2+ ions to calmodulin, its tryptic fragments TR1C and TR2C (which each constitute a globular domain of a pair of EF hands) and calbindin D9k. Macroscopic Ca2+ binding constants have been obtained over a range of ionic strengths (0 to 0.15 M KCl). For calmodulin the measurements indicate that the two separate globular domains TR1C and TR2C retain the Ca2+ binding properties they have in the intact molecule, with positive cooperativity within each domain. The absolute value of the free energy of interaction between the two sites in each domain, a measure of the cooperativity, increases with ionic strength and is greater than or equal to 10 kJ mol-1 at 0.15 M KCl. Two-dimensional 1H NMR studies show that the addition of KCl does not alter the conformation of the protein. In the case of calbindin D9k several categories of mutants have been studied. One group encompasses the effect of protein surface charges 5 to 15 A from the Ca2+ binding sites. Two-dimensional 1H NMR shows that neither the addition of KCl, nor mutations that neutralize the surface charges, change the protein conformation. Although the global structure of calbindin D9k is largely unchanged upon binding of calcium, the structure with only one cation bound is more similar to the (Ca2+)2 form. Interestingly, the dynamical properties of the Ca(2+)-free and the (Ca2+)2-forms of calbindin differ greatly. For example, the rate of NH/ND exchange of the Ca(2+)-free form is on average 200 times faster than that of the (Ca2+)2-form. The results obtained so far point to a non-negligible entropic contribution to the observed cooperativity of Ca2+ binding.

Amides↗
PubMed1990

1H NMR assignment and secondary structure of the Ca2(+)-free form of the amino-terminal epidermal growth factor like domain in coagulation factor X.

Blood coagulation factor X is composed of discrete domains, two of which are homologous to the epidermal growth factor (EGF). The N-terminal EGF like domain in factor X (fX-EGFN), residues 45-86 of the intact protein, contains a beta-hydroxylated aspartic acid and has one Ca2(+)-binding site. Using 2D NMR techniques, we have made a full assignment of the 500-MHz 1H NMR spectrum of Ca2(+)-free fX-EGFN. On the basis of this assignment and complementary NOESY experiments, we have also determined the secondary structure of Ca2(+)-free fX-EGFN in water solution. Residues 45-49 are comparatively mobile, whereas residues 50-56 are constrained by two disulfide bonds to one side of an antiparallel beta-sheet involving residues 59-64 and 67-72. Another antiparallel beta-sheet involves residues 76-77 and 83-84. A small, parallel beta-sheet connects residues 80-81 and 55-56 and thereby orients the two antiparallel beta-sheets relative to each other. Four beta-turns are identified, involving residues 50-53, 56-59, 64-67, and 73-76. Residues 78-82 adopt an extended bend structure. On the basis of secondary structure and the location of the three disulfide bonds, we find that Asp 46, Asp 48, and Hya 63 are sufficiently close to each other to form a Ca2(+)-binding site. However, the amino terminus of the Ca2(+)-free form of fX-EGFN is not part of a triple-stranded beta-sheet as in other EGF like peptides. Differences and similarities between fX-EFGN and murine EGF with respect to secondary structure and conformational shifts are discussed.

Amino Acid Sequence↗