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J Kördel

Publications and source records attributed to J Kördel.

24 records · Page 2Linked to original sources
PubMed1990

The rate and structural consequences of proline cis-trans isomerization in calbindin D9k: NMR studies of the minor (cis-Pro43) isoform and the Pro43Gly mutant.

The EF-hand calcium-binding protein, calbindin D9k, exists in solution in the calcium-loaded state, as a 1:3 equilibrium mixture of two isoforms, the result of cis-trans isomerism at the Gly42-Pro43 peptide bond [Chazin et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2195-2198]. Nuclear magnetic resonance (NMR) studies of the minor (cis-Pro43) isoform and the Pro43----Gly mutant are reported here. The rate of cis----trans isomerization at the Pro43 peptide bond in the wild-type protein was determined by line-shape analysis at elevated temperatures, using a sample in which all amino acids, except Ser and Val, were deuterated. The cis----trans rate is calculated to be 0.2 s-1 at 25 degrees C, corresponding to a free energy of activation, delta G, of 77 kJ/mol. The complete sequence-specific 1H NMR assignments of the cis-Pro43 isoform and the Pro43----Gly mutant in the calcium-loaded state have been obtained by using standard methods combined with comparisons to the previously assigned major (trans-Pro43) isoform. This has permitted detailed comparative analysis of 1H NMR chemical shifts, backbone scalar coupling constants, and nuclear Overhauser effects. The minor isoform has a global fold that is identical with that of the major isoform. Structural changes imposed by cis-trans isomerization at Pro43 are highly localized to the linker loop (containing Pro43) that joins the two EF hands. The Pro43----Gly mutant has a global fold that is identical with the wild-type protein, but does not exhibit conformational heterogeneity. Only very limited structural differences are observed between mutant and wild-type protein, and these are also highly localized to the linker loop. The ion-binding properties of the mutant, as determined by 43Ca and 113Cd NMR, are found to be very similar to the wild-type protein. These results provide crucial evidence that justifies the calculation of high-resolution three-dimensional structures of the Pro43Gly mutant, rather than of the conformationally heterogeneous wild-type protein.

Amino Acid Sequence↗
PubMed1990

Peptidyl-prolyl cis-trans isomerase does not affect the Pro-43 cis-trans isomerization rate in folded calbindin D9k.

The calcium-binding protein calbindin D9k has previously been shown to exist in two folded forms only differing in the proline cis-trans isomerism of the Gly-42-Pro-43 amide bond. This bond is located in a flexible loop connecting the two EF-hand Ca2+ sites. Calbindin D9k therefore constitutes a unique test case for investigating if the recently discovered enzyme peptidyl-prolyl cis-trans isomerase (PPIase) can affect the cis-trans exchange rate in a folded protein. The 1H NMR saturation transfer technique has been used to measure the rate of interconversion between the cis and trans forms of calbindin in the presence of PPIase (PPIase:calbindin concentration ratio 1:10) at 35 degrees C. No rate enhancement could be detected.

Amino Acid Isomerases↗
PubMed1989

Identification of an isoaspartyl linkage formed upon deamidation of bovine calbindin D9k and structural characterization by 2D 1H NMR.

Preparations of recombinant bovine calbindin D9k (r-calbindin) that appear homogeneous on SDS electrophoresis gels have been shown by isoelectric focusing to be mixtures of proteins differing in net charge. The production of two isoforms with increased negative charge occurs during a routine urea denaturation step and can be effectively suppressed by replacing this procedure with thermal denaturation. The two isoforms have been separated from the native protein by DEAE-Sephacel ion-exchange chromatography. Amino acid sequencing of tryptic peptide fragments and two-dimensional (2D) 1H NMR studies establish that the isoforms correspond to calbindin D9k deamidated at Asn56 and that the major product has an isoaspartate (beta-linked peptide) residue at this position. The minor deamidated component is found to have a normal Asp-Gly alpha-linkage. A detailed analysis of proton chemical shifts, phi backbone dihedral angles, and nuclear Overhauser effects indicates that the global conformation of r-calbindin is not perturbed upon deamidation and that all elements of secondary structure are intact. The Asp56 form is nearly identical with the intact protein, whereas the structure of the iso-Asp56 form is perturbed, predominantly in the polypeptide segment Lys55-Asp58. These studies demonstrate that 2D 1H NMR techniques can be used to identify and quantitate the two isoforms produced upon deamidation of a protein and to assess changes in the local and global conformation.

Amino Acids↗
PubMed1989

1H NMR sequential resonance assignments, secondary structure, and global fold in solution of the major (trans-Pro43) form of bovine calbindin D9k.

A wide range of two-dimensional 1H NMR experiments have been used to completely assign the 500-MHz 1H NMR spectrum of recombinant Ca2+-saturated bovine calbindin D9k (76 amino acids, Mr = 8500). In solution, calbindin D9k exists as an equilibrium mixture of isoforms with trans (75%) and cis (25%) isomers of the peptide bond at Pro43 [Chazin et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2195-2198], which results in two sets of 1H NMR signals from approximately half of the amino acids. The complete 1H NMR assignments for the major, trans-Pro43 isoform are presented here. By use of an integrated strategy for spin system identification, 62 of the 76 spin systems could be assigned to the appropriate residue type. Sequence-specific assignments were then obtained by the standard method. Secondary structure elements were identified on the basis of networks of sequential and medium-range nuclear Overhauser effects (NOEs), 3JHN alpha spin coupling constants, and the location of slowly exchanging amide protons. Four helical segments and a short beta-sheet between the two calcium binding loops are found. These elements of secondary structure and a few additional long-range NOEs provide the global fold. Good agreement is found between the solution and crystal structures of the minor A form of bovine calbindin D9k and between the solution structures of the minor A form of bovine calbindin D9k and intact porcine calbindin D9k.

Amino Acid Sequence↗
PubMed1989

Proline isomerism leads to multiple folded conformations of calbindin D9k: direct evidence from two-dimensional 1H NMR spectroscopy.

A complete analysis of calbindin D9k by two-dimensional 1H nuclear magnetic resonance spectroscopy has established the existence of two conformations for the folded protein in solution. Well-resolved major and minor resonances in a ratio of 3:1 are observed throughout the 1H NMR spectrum. Two-dimensional exchange experiments show that the major and minor species are related by an equilibrium process. Analysis of short proton-proton distances along the peptide backbone, identified by two-dimensional nuclear Overhauser effect spectroscopy, provides unambiguous evidence that the two forms of the folded protein differ only in the isomerization state of the peptide bond between Gly-42 and Pro-43. Cis-trans isomerism of Pro-43 is thereby directly identified as the cause of multiple conformations for the folded protein in solution. In addition, when Pro-43 is mutated to a glycine residue there is no indication of multiple conformations. These results provide evidence for the possibility of conformational heterogeneity in the native state of globular proteins.

Calbindins↗