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Journal Issue - Volume 9 Issue 11 (2000)

Abstract The ba3‐type cytochrome c oxidase from Thermus thermophilus is known as a two subunit enzyme. Deduced from the crystal structure of this enzyme, we discovered the presence of an additional transmembrane helix “subunit IIa” spanning the membrane. The hydrophobic N‐terminally blocked protein was isolated in high yield using high‐performance liquid chromatography. Its complete amino acid sequence was determined by a combination of...

Abstract We describe the design of Escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the Thermus thermophilus cytochrome c552 gene. Key features are (1) construction of a plasmid‐borne, chimeric cycA gene encoding an Escherichia coli‐compatible, N‐terminal signal sequence (MetLysIleSerIleTyrAlaThrLeu AlaAlaLeuSerLeuAlaLeuProAlaGlyAla) followed by the amino acid sequence of mature Thermus cytochrome c552; and (2)...

Abstract The crystal structure of the photoprotein obelin (22.2 kDa) from Obelia longissima has been determined and refined to 1.7 Å resolution. Contrary to the prediction of a peroxide, the noncovalently bound substrate, coelenterazine, has only a single oxygen atom bound at the C2‐position. The protein‐coelenterazine 2‐oxy complex observed in the crystals is photo‐active because, in the presence of calcium ion, bioluminescence emission within...

Abstract Calbindin D28k is a highly conserved Ca2+‐binding protein abundant in brain and sensory neurons. The 261‐residue protein contains six EF‐hands packed into one globular domain. In this study, we have reconstituted calbindin D28k from two fragments containing three EF‐hands each (residues 1–132 and 133–261, respectively), and from other combinations of small and large fragments. Complex formation is studied by ion‐exchange and...

Abstract Chaperonins cpn60/cpn10 (GroEL/GroES in Escherichia coli) assist folding of nonnative polypeptides. Folding of the chaperonins themselves is distinct in that it entails assembly of a sevenfold symmetrical structure. We have characterized denaturation and renaturation of the recombinant human chaperonin 10 (cpn10), which forms a heptamer. Denaturation induced by chemical denaturants urea and guanidine hydrochloride (GuHCl) ...

Abstract Peptide GFSKAELAKARAAKRGGY folds in an α‐helical conformation that is stabilized by formation of a hydrophobic staple motif and an N‐terminal capping box (Munoz V, Blanco FJ, Serrano L, 1995, Struct Biol 2:380–385). To investigate backbone and side‐chain internal motions within the helix and hydrophobic staple, residues F2, A5, L7, A8, and A10 were selectively 13C‐ and 15N‐enriched and NMR relaxation experiments were performed in water...

Abstract The DNA‐binding domain of the yeast heat shock transcription factor (HSF) contains a strictly conserved proline that is at the center of a kink. To define the role of this conserved proline‐centered kink, we replaced the proline with a number of other residues. These substitutions did not diminish the ability of the full‐length protein to support growth of yeast or to activate transcription, suggesting that the proline at...

Abstract The formation of the N‐terminal β‐hairpin of ubiquitin is thought to be an early event in the folding of this small protein. Previously, we have shown that a peptide corresponding to residues 1–17 of ubiquitin folds autonomously and is likely to have a native‐like hairpin register. To investigate the causes of the stability of this fold, we have made mutations in the amino acids at the apex of the turn. We find that in a...

Abstract Previous conformational analysis of 10‐residue linear peptides enabled us to identify some cross‐strand side‐chain interactions that stabilize β‐hairpin conformations. The stabilizing influence of these interactions appeared to be greatly reduced when the interaction was located at the N‐ and C‐termini of these 10‐residue peptides. To investigate the effect of the position relative to the turn of favorable interactions on...

Abstract The Escherichia coli RecA protein triggers both DNA repair and mutagenesis in a process known as the SOS response. The 81‐residue E. coli protein DinI inhibits activity of RecA in vivo. The solution structure of DinI has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of residual dipolar couplings, obtained in bicelle and phage media, supplemented with J couplings and a...

Abstract The SH3 domain, comprised of approximately 60 residues, is found within a wide variety of proteins, and is a mediator of protein–protein interactions. Due to the large number of SH3 domain sequences and structures in the databases, this domain provides one of the best available systems for the examination of sequence and structural conservation within a protein family. In this study, a large and diverse alignment of SH3...

Abstract We develop a protocol for estimating the free energy difference between different conformations of the same polypeptide chain. The conformational free energy evaluation combines the CHARMM force field with a continuum treatment of the solvent. In almost all cases studied, experimentally determined structures are predicted to be more stable than misfolded “decoys.” This is due in part to the fact that the Coulomb energy of...

Abstract We have modeled the structure of human lymphotactin (hLpnt), by homology modeling and molecular dynamics simulations. This chemokine is unique in having a single disulfide bond and a long C‐terminal tail. Because other structural classes of chemokines have two pairs of Cys residues, compared to one in Lpnt, and because it has been shown that both disulfide bonds are required for stability and function, the question arises...

Abstract Metabotropic glutamate receptors (mGluRs) belong to the family 3 of G‐protein‐coupled receptors. On these proteins, agonist binding on the extracellular domain leads to conformational changes in the 7‐transmembrane domains required for G‐protein activation. To elucidate the structural features that might be responsible for such an activation mechanism, we have generated models of the amino terminal domain (ATD) of type 4...

Abstract Presentation of peptides derived from endogenous proteins by class I major histocompatibility complex molecules is essential both for immunological self‐tolerance and induction of cytotoxic T‐cell responses against intracellular parasites. Despite frequent and diverse post‐translational modification of eukaryotic cell proteins, very few class I‐bound peptides with post‐translationally modified residues are known. Here we...

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