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Journal Issue - Volume 15 Issue 2 (February 2006)

Abstract To address the role of glycosylation on fibrillogenicity of amyloidogenic chicken cystatin, the consensus sequence for N‐linked glycosylation (Asn106‐Ile108 → Asn106‐Thr108) was introduced by site‐directed mutagenesis into the wild‐type and amyloidogenic chicken cystatins to construct the glycosylated form of chicken cystatins. Both the glycosylated and unglycosylated forms of wild‐type and amyloidogenic mutant I66Q cystatin were...

Abstract The biological activity of DnaK, the bacterial representative of the Hsp70 protein family, is regulated by the allosteric interaction between its nucleotide and peptide substrate binding domains. Despite the importance of the nucleotide‐induced cycling of DnaK between substrate‐accepting and releasing states, the heterotropic allosteric mechanism remains as yet undefined. To further characterize this mechanism, the...

  • The redox couple of the cytochrome c

  • Abel Schejter, Michael D. Ryan, Erica R. Blizzard, Chongyao Zhang, Emanuel Margoliash, Benjamin A. Feinberg
  • Published in Wiley Interscience on Jan 01, 2009
  • DOI: 10.1110/ps.051825906 (p 234-241)

Abstract Contrary to most heme proteins, ferrous cytochrome c does not bind ligands such as cyanide and CO. In order to quantify this observation, the redox potential of the ferric/ferrous cytochrome c–cyanide redox couple was determined for the first time by cyclic voltammetry. Its E0′ was −240 mV versus SHE, equivalent to −23.2 kJ/mol. The entropy of reaction for the reduction of the cyanide complex was also determined. From a thermodynamic...

Abstract Molecular chaperones of the Hsp70 family (bacterial DnaK, DnaJ, and GrpE) were shown to be strictly required for refolding of firefly luciferase from a denatured state and thus for effective restoration of its activity. At the same time the luciferase was found to be synthesized in an Escherichia coli cell‐free translation system in a highly active state in the extract with no chaperone activity. The addition of the chaperones ...

Abstract Regulation of programmed cell death by Bcl‐xL is dependent on both its solution and integral membrane conformations. A conformational change from solution to membrane is also important in this regulation. This conformational change shows a pH‐dependence similar to the translocation domain of diphtheria toxin, where an acid‐induced molten globule conformation in the absence of lipid vesicles mediates the change from solution...

Abstract Allosteric interactions between residues that are spatially apart and well separated in sequence are important in the function of multimeric proteins as well as single‐domain proteins. This observation suggests that, among the residues that are involved in long‐range communications, mutation at one site should affect interactions at a distant site. By adopting a sequence‐based approach, we present an automated approach that...

Abstract The nuclease domain of ColE7 (N‐ColE7) contains an H‐N‐H motif that folds in a ββα‐metal topology. Here we report the crystal structures of a Zn2+‐bound N‐ColE7 (H545E mutant) in complex with a 12‐bp duplex DNA and a Ni2+‐bound N‐ColE7 in complex with the inhibitor Im7 at a resolution of 2.5 Å and 2.0 Å, respectively. Metal‐dependent cleavage assays showed that N‐ColE7 cleaves double‐stranded DNA with a single metal ion cofactor, Ni2+,...

Abstract Glutathione S‐transferase of the malarial parasite Plasmodium falciparum (PfGST) represents a novel class of GST isoenzymes. Since the architecture of the PfGST substrate binding site differs significantly from its human counterparts and there is only this one isoenzyme present in the parasite, PfGST is considered a highly attractive target for antimalarial drug development. Here we report the mechanistic, kinetic, and structural...

Abstract The lymphocyte function‐associated antigen‐1 (LFA‐1) binding of a unique class of small‐molecule antagonists as represented by compound 3 was analyzed in comparison to that of soluble intercellular adhesion molecule‐1 (sICAM‐1) and A‐286982, which respectively define direct and allosteric competitive binding sites within LFA‐1's inserted (I) domain. All three molecules antagonized LFA‐1 binding to ICAM‐1‐Immunoglobulin G...

Abstract High hydrostatic pressure (HHP)‐mediated solubilization and refolding of five inclusion bodies (IBs) produced from bacteria, three Gram‐negative binding proteins (GNBP1, GNBP2, and GNBP3) from Drosophila, and two phosphatases from human were investigated in combination of a redox‐shuffling agent (2 mM DTT and 6 mM GSSG) and various additives. HHP (200 MPa) combined with the redox‐shuffling agent resulted in solubilization yields of...

Abstract Hepatoma Derived Growth Factor (HDGF) is an endogenous nuclear‐targeted mitogen that is linked with human disease. HDGF is a member of the weakly conserved PWWP domain family. This 70–amino acid motif, originally identified from the WHSC1 gene, has been found in more than 60 eukaryotic proteins. In addition to the PWWP domain, many proteins in this class contain known chromatin remodeling domains, suggesting a role for HDGF in...

Abstract A mutational analysis of the femtomolar‐affinity anti‐fluorescein antibody 4M5.3, compared to its wild‐type progenitor, 4‐4‐20, indicates both context‐dependent and ‐independent mutations are responsible for the 1800‐fold affinity improvement. 4M5.3 was engineered from 4‐4‐20 by directed evolution and contains 14 mutations. The seven mutations identified as present in each of 10 final round affinity maturation clones were...

Abstract Dynein light chain protein, a part of the cytoplasmic motor assembly, is a homodimer at physiological pH and dissociates below pH 4.5 to a monomer. The dimer binds to a variety of cargo, whereas the monomer does not bind any of the target proteins. We report here the pH induced stepwise structural and motional changes in the protein, as derived from line broadening and 15N transverse relaxation measurements. At pH 7 and...

Abstract Photonic induced immobilization is a novel technology that results in spatially oriented and spatially localized covalent coupling of biomolecules onto thiol‐reactive surfaces. Immobilization using this technology has been achieved for a wide selection of proteins, such as hydrolytic enzymes (lipases/esterases, lysozyme), proteases (human plasminogen), alkaline phosphatase, immunoglobulins' Fab fragment (e.g., antibody...

Abstract Proteins evolved through the shuffling of functional domains, and therefore, the same domain can be found in different proteins and species. Interactions between such conserved domains often involve specific, well‐determined binding surfaces reflecting their important biological role in a cell. To find biologically relevant interactions we developed a method of systematically comparing and classifying protein domain...

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