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R H Kretsinger

Publications and source records attributed to R H Kretsinger.

At least 37 records · Page 2Linked to original sources
PubMed1994

Crystal structure of human class mu glutathione transferase GSTM2-2. Effects of lattice packing on conformational heterogeneity.

The structures of three crystal forms of the class mu human glutathione transferase GSTM2-2 have been determined. X-ray phase information was obtained independently from molecular replacement and from anomalous scattering by a single isomorphous derivative. One crystal form contains a single monomer in the asymmetric unit and has been refined to 1.85 A with an overall R factor of 22.6%. The second form contains a single dimer in the asymmetric unit and has been refined to 3.5 A with an R factor of 20.7%. The third form contains two dimers in the asymmetric unit and has been refined to 3.0 A with an R factor of 25.0%. Although all three crystal forms were grown from solutions that contained glutathione-dinitrobenzene, electron density can only be seen for the glutathione portion of the ligand. The first 202 residues in the seven crystallographically independent monomers of GSTM2-2 are essentially identical in structure. However, heterogeneity in the conformation of the side-chain of Tyr115 is observed in the different monomers. The tertiary structure of residues 1-202 is similar to that of the corresponding region in the class mu isoform of glutathione transferase from rat, GST3-3 (Ji et al. (1992), Biochemistry, 31, 10169-10184). However, significant differences in the conformation of the two enzymes have been observed in the region of the active site that binds hydrophobic substrates. These differences include a 2 A shift in the carboxy terminus of a helix, and significant heterogeneity in the conformation of the last 15 residues of the carboxy terminus. The conformation and degree of disorder of the last 15 residues correlates with the extent of protein-protein contacts within the unit cell.

Amino Acid Sequence↗
PubMed1994

p-Chlorotetrafluorophenyl esters of N-protected amino acids.

p-Chlorotetrafluorophenyl (Tfc) esters of protected amino acids and peptides are more reactive than are the well known pentafluorophenyl (Pfp) esters. Two reagents, p-chlorotetrafluorophenyltrifluoroacetate (Tfc-OTfa) and di-(p-chlorotetrafluorophenyl)carbonate (di-Tfc-carbonate), can be used for their syntheses, thereby avoiding use of the allergic dicyclohexylcarbodiimide. This is especially important for bulk preparations. Many Fmoc- and Boc-amino acid-OTfc esters have been synthesized and characterized. The hexadecameric tandem repeat H-(AlaAlaLysPro)4-OH was synthesized using di-Tfc-carbonate for the preparation of Tfc-esters.

Alanine↗
PubMed1993

The linker of des-Glu84-calmodulin is bent.

The crystal structure of a mutant calmodulin (CaM) lacking Glu-84 has been refined to R = 0.23 using data measured to 2.9-A resolution. In native CaM the central helix is fully extended, and the molecule is dumbbell shaped. In contrast, the deletion of Glu-84 causes a bend of 95 degrees in the linker region of the central helix at Ile-85. However, EF-hand domains 1 and 2 (lobe 1,2) do not touch lobe 3,4. The length, by alpha-carbon separation, of des-Glu84-CaM is 56 A; that of native CaM is 64 A. The shape of des-Glu84-CaM is similar to that of native CaM, as it is bound to the target peptide of myosin light-chain kinase. This result supports the proposal that the linker region of the central helix of CaM functions as a flexible tether.

Amino Acid Sequence↗
PubMed1993

Otoconin-22, the major protein of aragonitic frog otoconia, is a homolog of phospholipase A2.

Otoconia are composites of proteins and inorganic crystals formed in the peripheral portion of the vestibular system of vertebrates. They add mass to the extracellular otoconial membrane, thereby increasing its deflection during linear acceleration. This added mass increases the sensitivity of the underlying sensory maculae. Otoconia provide a promising system to decipher the interaction of protein and mineral during the growth and maintenance of biominerals. We have purified the major protein of the aragonitic otoconia of Xenopus laevis, which we call otoconin-22, and determined its amino acid sequence and carbohydrate composition. The 127 residues are 37% identical to the phospholipase A2 from Crotalus atrox. We propose that otoconin-22 from X. laevis is homologous to phospholipase A2 and has a similar tertiary structure.

Amino Acid Sequence↗
PubMed1993

Evolution of EF-hand calcium-modulated proteins. III. Exon sequences confirm most dendrograms based on protein sequences: calmodulin dendrograms show significant lack of parallelism.

In the first report in this series we presented dendrograms based on 152 individual proteins of the EF-hand family. In the second we used sequences from 228 proteins, containing 835 domains, and showed that eight of the 29 subfamilies are congruent and that the EF-hand domains of the remaining 21 subfamilies have diverse evolutionary histories. In this study we have computed dendrograms within and among the EF-hand subfamilies using the encoding DNA sequences. In most instances the dendrograms based on protein and on DNA sequences are very similar. Significant differences between protein and DNA trees for calmodulin remain unexplained. In our fourth report we evaluate the sequences and the distribution of introns within the EF-hand family and conclude that exon shuffling did not play a significant role in its evolution.

Animals↗
PubMed1993

Evolution of EF-hand calcium-modulated proteins. IV. Exon shuffling did not determine the domain compositions of EF-hand proteins.

In the previous three reports in this series we demonstrated that the EF-hand family of proteins evolved by a complex pattern of gene duplication, transposition, and splicing. The dendrograms based on exon sequences are nearly identical to those based on protein sequences for troponin C, the essential light chain myosin, the regulatory light chain, and calpain. This validates both the computational methods and the dendrograms for these subfamilies. The proposal of congruence for calmodulin, troponin C, essential light chain, and regulatory light chain was confirmed. There are, however, significant differences in the calmodulin dendrograms computed from DNA and from protein sequences. In this study we find that introns are distributed throughout the EF-hand domain and the interdomain regions. Further, dendrograms based on intron type and distribution bear little resemblance to those based on protein or on DNA sequences. We conclude that introns are inserted, and probably deleted, with relatively high frequency. Further, in the EF-hand family exons do not correspond to structural domains and exon shuffling played little if any role in the evolution of this widely distributed homolog family. Calmodulin has had a turbulent evolution. Its dendrograms based on protein sequence, exon sequence, 3'-tail sequence, intron sequences, and intron positions all show significant differences.

Animals↗
PubMed1993

Inferred protein content and distribution from density measurements of calcitic and aragonitic otoconia.

Otoconia from the peripheral portion of the vestibular system contain specific proteins and are mineralized by several polymorphs of calcium carbonate. To infer the internal distribution of their mineral and protein components, we have measured the densities, by equilibrium centrifugation, and the lengths of aragonitic otoconia from the African clawed frog (Xenopus laevis) and calcitic otoconia from the Norway rat (Rattus norvegicus). The densities are statistically dependent on the length of the otoconia. Further, this dependence is not the same for aragonitic and calcitic otoconia. Aragonitic otoconia are statistically more dense when smaller and less dense when larger. For calcitic otoconia the opposite is true; the smaller otoconia are statistically less dense than larger otoconia. Because the organic and inorganic phases have different densities, this indicates that the otoconial proteins and the mineral phases have different distributions within these two types of otoconia.

Animals↗
PubMed1992

Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories.

In the first report in this series we described the relationships and evolution of 152 individual proteins of the EF-hand subfamilies. Here we add 66 additional proteins and define eight (CDC, TPNV, CLNB, LPS, DGK, 1F8, VIS, TCBP) new subfamilies and seven (CAL, SQUD, CDPK, EFH5, TPP, LAV, CRGP) new unique proteins, which we assume represent new subfamilies. The main focus of this study is the classification of individual EF-hand domains. Five subfamilies--calmodulin, troponin C, essential light chain, regulatory light chain, CDC31/caltractin--and three uniques--call, squidulin, and calcium-dependent protein kinase--are congruent in that all evolved from a common four-domain precursor. In contrast calpain and sarcoplasmic calcium-binding protein (SARC) each evolved from its own one-domain precursor. The remaining 19 subfamilies and uniques appear to have evolved by translocation and splicing of genes encoding the EF-hand domains that were precursors to the congruent eight and to calpain and to SARC. The rates of evolution of the EF-hand domains are slower following formation of the subfamilies and establishment of their functions. Subfamilies are not readily classified by patterns of calcium coordination, interdomain linker stability, and glycine and proline distribution. There are many homoplasies indicating that similar variants of the EF-hand evolved by independent pathways.

Amino Acid Sequence↗
PubMed1992

Identification of a new EF-hand superfamily member from Trypanosoma brucei.

We identified several open reading frames between the regions encoding calmodulin and ubiquitin-EP52/1 in the genome of Trypanosoma brucei. One of these, EFH5, encodes a protein 192 amino acids long. The EFH5 transcript is present in poly(A)+ mRNA and is present at similar levels in the mammalian bloodstream form and the insect procyclic form. EFH5 contains four EF-hand homolog domains, two of which are inferred to bind Ca2+ ions. We expressed EFH5 as a fusion protein in Escherichia coli and demonstrated calcium-binding activity of the fusion protein using the 45Ca-overlay technique. The function of EFH5 remains unknown; however, as the fourth EF-hand homolog identified in trypanosomes, it attests to the broad range of functions assumed by calcium functioning as a second messenger. EFH5, which is most closely related to LAV1-2 from Physarum, represents a distinct subfamily among the EF-hand-containing proteins.

Amino Acid Sequence↗
PubMed1991

Small-angle X-ray scattering studies of calmodulin mutants with deletions in the linker region of the central helix indicate that the linker region retains a predominantly alpha-helical conformation.

Two mutant forms of calmodulin were examined by small-angle X-ray scattering in solution and compared with the wild-type protein. Each mutant has deletions in the linker region of the central helix: one lacks residues Glu-83 and Glu-84 (Des2) and the other lacks residues Ser-81 through Glu-84 (Des4). The deletions change both the radii of gyration and the maximum dimensions of the molecules. In the presence of Ca2+, the observed radii of gyration are 22.4 A for wild-type bacterially expressed calmodulin, 19.5 A for Des2 calmodulin, and 20.3 A for Des4 calmodulin. A reduction in the radius of gyration by 1-2 A on removal of calcium, previously observed in the native protein, was also found in the wild type and the Des4 mutant; however, no significant size change was observed in the Des2 mutant. The large calcium-dependent conformational change in calmodulin induced by the binding of melittin [Kataoka, M., Head, J.F., Seaton, B.A., & Engelman, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6944-6948] was observed in all the bacterially expressed proteins. Each protein appears to undergo a transition from a dumbbell shape to a more globular conformation on binding melittin in the presence of calcium, although quantitatively the changes in the wild-type and Des4 proteins greatly exceed those in Des2. Modeling shows the central linker region of the molecule. Thus, the structure of the linker region is stable enough to maintain the average orientation and separation of the lobes yet flexible enough to permit the lobes to approach each other upon binding a peptide.

Amino Acid Sequence↗
PubMed1991

Trifluoperazine binding to mutant calmodulins.

Trifluoperazine (TFP) binding by 14 calmodulins, including 12 produced by site-directed mutagenesis, was determined. While vertebrate calmodulin binds 4.2 +/- 0.2 equiv of TFP, Escherichia coli expressed but unmutated calmodulins bind about 5.0 +/- 0.5 equiv of TFP. The cause for this difference is not known. The E. coli expressed proteins consist of two different series expressed from different calmodulin genes, CaMI and SYNCAM. The wild-type genes code for proteins that differ by nine conservative amino acid substitutions. Both these calmodulins bind 5 equiv of TFP with similar affinities, thus none of these conservative substitutions has any additional effect on TFP binding. Some altered calmodulins (deletion of EE83-84 or SEEE81-84, changing DEE118-120----KKK, M124----I,E120----K, or E82----K) have no appreciable effect on TFP binding. Other mutations affect either the binding of one TFP (deletion of E84) or about two TFP (changing E84----K, EEE82-84----KKK, E67----A, DEQ6-8----KKK, or E11----K). The mutations that affect TFP binding are localized to three regions of calmodulin: The amino-terminal alpha-helix, the central helix between the two globular ends of calmodulin, and a calcium-binding site in the second calcium-binding domain. The results are consistent with each of these regions either directly participating in drug binding or involved structurally in maintaining or inducing the correct conformation for TFP binding in the amino-terminal half of calmodulin.

Amino Acid Sequence↗
PubMed1991

Calmodulins with deletions in the central helix functionally replace the native protein in yeast cells.

Deletion of Glu-84, Glu-83 and Glu-84, or Ser-Glu-Glu-Glu (residues 81-84) from the central helix of mammalian calmodulin is known to result in a 5-7 times decrease in its apparent in vitro affinities for three calmodulin-dependent enzymes. However, based on in vitro experiments, it is difficult to estimate how these deletions might affect in vivo cellular function. The yeast Saccharomyces cerevisiae, which requires calmodulin for growth, provides an excellent system to evaluate these deletion proteins in vivo. Based on its ability to restore normal growth characteristics to yeast cells, mammalian calmodulin is functionally identical to the yeast protein; herein we evaluate the effect of deleting residues 84, 83 and 84, or 81-84 from the central helix. Sequences encoding the deletion proteins and an unaltered control sequence were introduced by means of a yeast shuttle vector and were expressed under control of the yeast calmodulin promoter. The deletion and control calmodulins are produced at levels similar to that observed for the yeast protein, and they completely restore normal growth characteristics. This result suggests that the regions deleted from the central helix are not critical for activation of any yeast calmodulin target normally required for cell growth or division. It is likely that there are twisting and shortening motions associated with the deletions from the central helix that alter significantly the spatial relationship between the two lobes of calmodulin. The abilities of the deletion calmodulins to restore completely normal growth characteristics to yeast cells suggest that the lobes of all the deletion proteins can still be appropriately positioned in calmodulin-target complexes. This is consistent with the hypothesis that the central helix of calmodulin is analogous to a flexible tether rather than to a rigid connector between the two lobes of the molecule.

Amino Acid Sequence↗
PubMed1991

Noncovalent complex between domain AB and domains CD*EF of parvalbumin.

The interaction between domain AB and domains CD*EF of pike parvalbumin III has been studied by intrinsic fluorescence spectroscopy. In the presence of Ca2+ ions, parvalbumin fragment 38-108 containing two calcium binding sites interacts with the short peptide 1-37 with association constant 10(5.3 +/- 0.5) M-1. Removal of Ca2+ ions results in the disappearance of the interaction. The affinity of the complex of the two fragments for calcium is 50-times higher than the affinity of the isolated fragment 38-108, but slightly lower than that of the intact protein.

Binding Sites↗
PubMed1991

Microcrystals of the annexin, p68: paracrystal to crystal transition and molecular packing as determined by electron microscopy and image reconstruction.

The calcium-sensitive, membrane-binding annexin, p68, has been crystallized from solutions of polyethylene glycol and ammonium sulfate. Our electron microscopy and X-ray diffraction data indicate that p68 crystals are tetragonal, in space group P4(1), and have unit cell dimensions of a = b = 68.4 A and c = 209.6 A. The mechanism of crystallization from polyethylene glycol involves a transition from a paracrystalline form to ordered crystals by lateral reordering of chains of molecules extended along the c axis. These chains are directional and might reflect a mechanism whereby the two different ends of (chains of) the p68 molecules interact with different membranes.

Animals↗