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Journal Issue - Volume 9 Issue 7 (2000)

Abstract On the basis of crystal structures of the pyrroloquinoline quinone (PQQ) dependent enzymes methanol dehydrogenase (MDH) and soluble glucose dehydrogenase (s‐GDH), different catalytic mechanisms have been proposed. However, several lines of biochemical and kinetic evidence are strikingly similar for both enzymes. To resolve this discrepancy, we have compared the structures of these enzymes in complex with their natural...

Abstract Members of the serpin family of serine proteinase inhibitors play important roles in the inflammatory, coagulation, fibrinolytic, and complement cascades. An inherent part of their function is the ability to undergo a structural rearrangement, the stressed (S) to relaxed (R) transition, in which an extra strand is inserted into the central A β‐sheet. In order for this transition to take place, the A sheet has to be...

Abstract The ligand‐binding domain of the human low‐density lipoprotein receptor consists of seven modules, each of 40–45 residues. In the presence of calcium, these modules adopt a common polypeptide fold with three conserved disulfide bonds. A concatemer of the first and second modules (LB1‐2) folds efficiently in the presence of calcium ions, forming the same disulfide connectivities as in the isolated modules. The...

Abstract The active sites and substrate bindings of Rhizobium trifolii molonyl‐CoA synthetase (MCS) catalyzing the malonyl‐CoA formation from malonate and CoA have been determined based on NMR spectroscopy, site‐directed mutagenesis, and comparative modeling methods. The MCS‐bound conformation of malonyl‐CoA was determined from two‐dimensional‐transferred nuclear Overhauser effect spectroscopy data. MCS protein folds into two structural...

Abstract Previously, we determined the DNA and amino acid sequences as well as biochemical and biophysical properties of a series of fungal phytases. The amino acid sequences displayed 49‐68% identity between species, and the catalytic properties differed widely in terms of specific activity, substrate specificity, and pH optima. With the ultimate goal to combine the most favorable properties of all phytases in a single protein, we...

Abstract We present a model for the skeletal muscle troponin‐C (TnC)/troponin‐I (TnI) interaction, a critical molecular switch that is responsible for calcium‐dependent regulation of the contractile mechanism. Despite concerted efforts by multiple groups for more than a decade, attempts to crystallize troponin‐C in complex with troponin‐I, or in the ternary troponin complex, have not yet delivered a high‐resolution structure. Many...

Abstract We previously reported the de novo design of an amphiphilic peptide [YGG(IEKKIEA)4] that forms a native‐like, parallel triple‐stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile 18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple‐stranded α‐helix structure. The addition of Cd(II),...

Abstract Little is known of the plant branched‐chain α‐ketoacid dehydrogenase complex. We have undertaken a detailed study of the structure of the dihydrolipoyl acyltransferase (BCE2) subunit that forms the core of the complex, to which two other enzymes attach. Mature Arabidopsis thaliana BCE2 was expressed in Escherichia coli. The soluble recombinant protein was purified using a Superose 6 size‐exclusion column to >90% homogeneity and was...

Abstract The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 °C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 °C, the hydrophobic interaction should make only a small...

Abstract Bovine β‐lactoglobulin (BLG) in vivo has been found complexed with fatty acids, especially palmitic and oleic acid. To elucidate the still unknown structure‐function relationship in this protein, the interactions between 13C enriched palmitic acid (PA) and BLG were investigated by means of one‐, two‐, and three‐dimensional NMR spectroscopy in the pH range 8.4‐2.1. The NMR spectra revealed that at neutral pH the ligand is bound within...

  • Autopalmitoylation of tubulin

  • J. Wolff, Anna Maria Zambito, P. Jeram Britto, Leslie Knipling
  • Published in Wiley Interscience on Dec 31, 2008
  • DOI: 10.1110/ps.9.7.1357 (p 1357-1364)

Abstract Pure rat brain tubulin is readily palmitoylated in vitro using [3H]palmitoyl CoA but no added enzymes. A maximum of approximately six palmitic acids are added per dimer in 2–3 h at 36–37 °C under native conditions. Both α and β tubulin are labeled, and 63–73% of the label was hydroxylamine‐labile, presumed thioesters. Labeling increases with increasing pH and temperature, and with low concentrations of guanidine HCl or KCl (but not...

Abstract The disulfide bond pattern of catrocollastatin‐C was determined by N‐terminal sequencing and mass spectrometry. The N‐terminal disintegrin‐like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin‐like...

Abstract Eucaryotes possess one or more NADP‐dependent methylene‐THF dehydrogenases as part of multifunctional enzymes. In addition, yeast expresses an unusual monofunctional NAD‐dependent enzyme, yMTD. We report X‐ray structures for the apoenzyme and its complex with NAD+ at 2.8 and 3.0 Å resolution, respectively. The protein fold resembles that seen for the human and Escherichia coli dehydrogenase/cyclohydrolase bifunctional enzymes. The...

Abstract Domains belonging to the immunoglobulin‐like fold are responsible for a wide variety of molecular recognition processes. Here we describe a new family of domains, the HYR family, which is predicted to belong to this fold, and which appears to be involved in cellular adhesion. HYR domains were identified in several eukaryotic proteins, often associated with Complement Control Protein (CCP) modules or arranged in multiple...

Abstract Six helix surface positions of protein G (Gβ1) were redesigned using a computational protein design algorithm, resulting in the five fold mutant Gβ1m2. Gβ1m2 is well folded with a circular dichroism spectrum nearly identical to that of Gβ1, and a melting temperature of 91 °C, ∼6°C higher than that of Gβ1. The crystal structure of Gβ1m2 was solved to 2.0 Å resolution by molecular replacement. The absence of hydrogen bond or salt bridge...

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