Biomedical subjects

R Ficner

Publications and source records attributed to R Ficner.

24 records · Page 2Linked to original sources

PubMed1995

Isolation, crystallization, crystal structure analysis and refinement of allophycocyanin from the cyanobacterium Spirulina platensis at 2.3 A resolution.

The phycobiliprotein allophycocyanin from the cyanobacterium Spirulina platensis has been isolated and crystallized. The crystals belong to space group P6(3)22 with cell constants a = b = 101.9 A, c = 130.6 A, alpha = beta = 90 degrees, gamma = 120 degrees, with one (alpha beta) monomer in the asymmetric unit. The three-dimensional structure of the (alpha beta) monomer was solved by multiple isomorphous replacement. The crystal structure has been refined in a cyclic manner by energy-restrained crystallographic refinement and model building. The conventional crystallographic R-factor of the final model is 19.6% with data from 8.0 to 2.3 A. The molecular structure of the subunits resembles other solved phycobiliprotein structures. In comparison to C-phycocyanin and b-phycoerythrin the major differences arise from deletions and insertions of segments involved in the protein-chromophore interactions. The stereochemistry of the alpha 84 and beta 84 chiral atoms are C(2)-R, C(3)-R and C(31)-R. The configuration (C(4)-Z, C(10)-Z and C(15)-Z) and the conformation (C(5)-anti, C(9)-syn and C(14)-anti) are equal for both chromophores.

Amino Acid Sequence↗

PubMed1995

Three-dimensional structure of the bifunctional protein PCD/DCoH, a cytoplasmic enzyme interacting with transcription factor HNF1.

The bifunctional protein pterin-4a-carbinolamine dehydratase (PCD)/dimerization cofactor of HNF1 (DCoH) is a cytoplasmic enzyme involved in the tetrahydrobiopterin regeneration and is found in complex with the transcription factor HNF1 in liver cell nuclei. An atypical hyperphenylalaninemia and the depigmentation disorder vitiligo are related to a deficiency of PCD/DCoH activity. The crystal structure of PCD/DCoH was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 20.5% at 2.7 A resolution. The single domain monomer comprises three alpha-helices packed against one side of a four-stranded, antiparallel beta-sheet. The functional enzyme is a homo-tetramer of 222 symmetry where each of the monomers contributes one helix to a central four helix bundle. In the tetramer two monomers form an eight-stranded, antiparallel beta-sheet with six helices packing against it from one side. The concave, hydrophobic surface of the eight-stranded beta-sheet with its two protruding loops at either end is reminiscent of the saddle-like shape seen in the TATA-box binding protein. PCD/DCoH binds as a dimer to the helical dimerization domain of dimeric HNF1 forming a hetero-tetramer possibly through a mixed four helix bundle.

Amino Acid Sequence↗

PubMed1995

Crystallization and preliminary crystallographic studies of recombinant dimerization cofactor of transcription factor HNF1/pterin-4 alpha-carbinolamine dehydratase from liver.

The bi-functional protein dimerization cofactor of HNF1 (DCoH)/pterin-4 alpha-carbinolamine dehydratase (PCD) is found in liver cell nuclei bound to the transcription factor hepatocyte nuclear factor 1 (HNF1) as well as in the cytoplasm acting as an enzyme involved in the phenylalanine hydroxylation system. Deficiency of DCoH/PCD activity in liver causes an atypical hyperphenylalaninemia and deficiency in human epidermis is related to the depigmentation disorder vitiligo. DCoH/PCD from rat liver, which is identical to the human protein, was expressed in E. coli, purified to homogeneity and crystallized. The crystals belong to the trigonal space group P3(1)21 (or P3(2)21) with unit cell dimensions of a = b = 106.2 A, c = 197.1 A. Native crystals diffract to a resolution of 2.5 A.

Animals↗

PubMed1995

Sequence analysis and overexpression of the Zymomonas mobilis tgt gene encoding tRNA-guanine transglycosylase: purification and biochemical characterization of the enzyme.

tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q). It catalyzes the posttranscriptional base exchange of the Q precursor 7-aminomethyl-7-deazaguanine (preQ1) with the genetically encoded guanine in the anticodon of tRNA(Asp), tRNA(Asn), tRNA(His), and tRNA(Tyr). A partially sequenced gene upstream of the DNA ligase (lig) gene of the Zymomonas mobilis chromosome shows strong homology to the tgt gene of Escherichia coli (K.B. Shark and T. Conway, FEMS Microbiol. Lett. 96:19-26, 1992). We showed that this gene is able to complement the tgt mutation in E. coli SJ1505, and we determined its complete sequence. Four start codons were possible for this gene, resulting in proteins of 386 to 399 amino acids (M(r), 42,800 to 44,300) showing 60.4% sequence identity with Tgt from E. coli. The smallest of the four possible reading frames, which was still extended at its 5' end compared with the E. coli tgt gene, was overexpressed in E. coli. The gene product was purified to homogeneity and was biochemically characterized. The kinetical parameters were virtually identical to those published for the E. coli enzyme. In contrast to E. coli Tgt, which is reported to be a homotrimer, Z. mobilis Tgt was found to be a monomer according to gel filtration. In this study, it was shown that the formation of homotrimers by the E. coli enzyme is readily reversible and is dependent on protein concentration.

Amino Acid Sequence↗

PubMed1993

Refined crystal structure of phycoerythrin from Porphyridium cruentum at 0.23-nm resolution and localization of the gamma subunit.

The three-dimensional structure of the light-harvesting pigment-protein b-phycoerythrin from the red alga Porphyridium cruentum has been determined at 0.23-nm resolution. The b-phycoerythrin structure is very similar to the structure of B-phycoerythrin from Porphyridium sordidum. Besides three non-identical residues there are only small differences between b-phycoerythrin and B-phycoerythrin alpha and beta subunits, respectively. In the crystals b-phycoerythrin forms an (alpha beta)6 hexamer (molecular mass: 236 kDa), whereas B-phycoerythrin additionally contains a 30-kDa gamma subunit. The comparison of the b-phycoerythrin and B-phycoerythrin electron-density maps clearly reveals, that the gamma subunit is located inside the (alpha beta)6 aggregate.

Crystallography, X-Ray↗

PubMed1992

Isolation, crystallization, crystal structure analysis and refinement of B-phycoerythrin from the red alga Porphyridium sordidum at 2.2 A resolution.

The light-harvesting pigment-protein complex B-phycoerythrin from the red alga Porphyridium sordidum has been isolated and crystallized. B-Phycoerythrin consists of three different subunits forming an (alpha beta)6 gamma aggregate. The three-dimensional structure of the (alpha beta)6 hexamer was solved by Patterson search techniques using the molecular model of C-phycocyanin from Fremyella diplosiphon. The asymmetric unit of the crystal cell (space group P3, with a = b = 111.2 A, c = 59.9 A, alpha = beta = 90 degrees, gamma = 120 degrees) contains two (alpha beta) monomers related by a local dyad. Three asymmetric units are arranged around the crystallographic 3-fold axis building an (alpha beta)6 hexamer, as in C-phycocyanin. The crystal structure has been refined by energy-restrained crystallographic refinement and model building. The conventional R-factor of the final model was 18.9% with data to 2.2 A resolution. The molecular structures of the alpha and beta-subunits resemble those of C-phycocyanin. Major changes in comparison to phycocyanin are caused by deletion or insertion of segments involved in protein-chromophore interactions. The singly linked phycoerythrobilin chromophores alpha-84, alpha-140a, beta-84 and beta-155 are each covalently bound to a cysteine by ring A. The doubly linked chromophore beta-50/beta-61 is attached at cysteine beta-50 through ring A and at cysteine beta-61 through ring D. B-Phycoerythrin contains additionally a 30 kDa gamma-subunit, which is presumably located in the central cavity of the hexamer. It is disordered, as a consequence of crystal and local symmetry averaging.

Amino Acid Sequence↗