The Experts below are selected from a list of 327 Experts worldwide ranked by ideXlab platform
Alexander Kozik - One of the best experts on this subject based on the ideXlab platform.
-
MECHANISM OF LIGAND-PROTEIN Interaction IN PLANT SEED THIAMINE-BINDING PROTEINS. PRELIMINARY CHEMICAL IDENTIFICATION OF AMINO ACID RESIDUES ESSENTIAL FOR THIAMINE BINDING TO THE BUCKWHEAT-SEED PROTEIN
Biochimie, 1996Co-Authors: Maria Rapala-kozik, Alexander KozikAbstract:Abstract Thiamine-binding protein, isolated from buckwheat seeds, was chemically modified in an attempt to identify amino acid residues involved in protein-thiamine Interaction. No evidence was found in support of specific roles of arginine residues, sulfhydryl groups, amino groups and tyrosine residues. Under carefully controlled reaction conditions (Tris pH 5–6), the modification with I-ethyl-3-(3-dimethylaminopropyl)carbodiimide caused a complete loss of thiamine-binding capacity. Thus, the carboxyl groups seemed to be essential for binding, possibly for Ionic Interaction with protein-bound thiamine cation. A selective modification of histidine residues using diethylpyrocarbonate correlated with a loss of thiamine-binding capacity; the modification and the loss of binding capacity could be reversed with hydroxylamine; some ligand-protection against modification was observed. From Tsou analysis of diethylpyrocarbonate modification and resulting loss of thiamine-binding it was suggested that 1–2 of 20 histidine residues of the protein were essential for thiamine binding. The essential histidine(s) might be present in the binding site and possibly were involved in hydrogen bonding(s) with protein-bound thiamine molecule.
Youhei Yamagata - One of the best experts on this subject based on the ideXlab platform.
-
Analysis of the Ionic Interaction between the hydrophobin RodA and two cutinases of Aspergillus nidulans obtained via an Aspergillus oryzae expression system.
Applied microbiology and biotechnology, 2016Co-Authors: Takumi Tanaka, Kei Nanatani, Toru Takahashi, Mayumi Nakayama, Youhei Yamagata, Keietsu AbeAbstract:Hydrophobins are amphipathic secretory proteins with eight conserved cysteine residues and are ubiquitous among filamentous fungi. In the fungus Aspergillus oryzae, the hydrophobin RolA and the polyesterase CutL1 are co-expressed when the sole available carbon source is the biodegradable polyester polybutylene succinate-co-adipate (PBSA). RolA promotes the degradation of PBSA by attaching to the particle surface, changing its structure and interacting with CutL1 to concentrate CutL1 on the PBSA surface. We previously reported that positively charged residues in RolA and negatively charged residues in CutL1 are cooperatively involved in the Ionic Interaction between RolA and CutL1. We also reported that hydrophobin RodA of the model fungus Aspergillus nidulans, which was obtained via an A. oryzae expression system, interacted via Ionic Interactions with CutL1. In the present study, phylogenetic and alignment analyses revealed that the N-terminal regions of several RolA orthologs contained positively charged residues and that the corresponding negatively charged residues on the surface of CutL1 that were essential for the RolA–CutL1 Interaction were highly conserved in several CutL1 orthologs. A PBSA microparticle degradation assay, a pull-down assay using a dispersion of Teflon particles, and a kinetic analysis using a quartz crystal microbalance revealed that recombinant A. nidulans RodA interacted via Ionic Interactions with two recombinant A. nidulans cutinases. Together, these results imply that Ionic Interactions between hydrophobins and cutinases may be common among aspergilli and other filamentous fungi.
-
Ionic Interaction of positive amino acid residues of fungal hydrophobin rola with acidic amino acid residues of cutinase cutl1
Molecular Microbiology, 2015Co-Authors: Toru Takahashi, Takumi Tanaka, Youhei Yamagata, Yusei Tsushima, Kimihide Muragaki, Kenji Uehara, Shunsuke Takeuchi, Hiroshi MaedaAbstract:Hydrophobins are amphipathic proteins secreted by filamentous fungi. When the industrial fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate co-adipate (PBSA), it produces RolA, a hydrophobin, and CutL1, a PBSA-degrading cutinase. Secreted RolA attaches to the surface of the PBSA particles and recruits CutL1, which then condenses on the particles and stimulates the hydrolysis of PBSA. Here, we identified amino acid residues that are required for the RolA-CutL1 Interaction by using site-directed mutagenesis. We quantitatively analyzed kinetic profiles of the Interactions between RolA variants and CutL1 variants by using a quartz crystal microbalance (QCM). The QCM analyses revealed that Asp142, Asp171 and Glu31, located on the hydrophilic molecular surface of CutL1, and His32 and Lys34, located in the N-terminus of RolA, play crucial roles in the RolA-CutL1 Interaction via Ionic Interactions. RolA immobilized on a QCM electrode strongly interacted with CutL1 (K(D) = 6.5 nM); however, RolA with CutL1 variants, or RolA variants with CutL1, showed markedly larger KD values, particularly in the Interaction between the double variant RolA-H32S/K34S and the triple variant CutL1-E31S/D142S/D171S (K(D) = 78.0 nM). We discuss a molecular prototype model of hydrophobin-based enzyme recruitment at the solid-water interface.
Torgny Låås - One of the best experts on this subject based on the ideXlab platform.
-
Size-exclusion chromatography of DNA restriction fragments : Fragment length determinations and a comparison with the behaviour of proteins in size-exclusion chromatography
Journal of Chromatography A, 2002Co-Authors: Hans Ellegren, Torgny LååsAbstract:Abstract Size-exclusion chromatography of double-stranded DNA restriction fragments on Superose 6® is shown to be an accurate method for chain length determination of unknown DNA. Ionic Interaction was observed between DNA and the gel matrix but was easily prevented by the addition of 0.15–0.2 M sodium chloride to the eluent. Compared to protein, the selectivity curve of DNA fragments was found to be steeper reflecting the different chromatographic behaviour of rod-like and globular molecules. The relationships between the selectivity curves of DNA and protein were similar on Superose 6 and on Sephacryl® S-500.
Peter A. Rubenstein - One of the best experts on this subject based on the ideXlab platform.
-
importance of a lys113 glu195 intermonomer Ionic bond in f actin stabilization and regulation by yeast formins bni1p and bnr1p
Journal of Biological Chemistry, 2013Co-Authors: Melissa Mckane, Peter A. RubensteinAbstract:Abstract Proper actin cytoskeletal function requires actin's ability to generate a stable filament and requires that this reaction be regulated by actin-binding proteins via allosteric effects on the actin. A proposed Ionic Interaction in the actin filament interior between Lys113 of one monomer and Glu195 of a monomer in the apposing strand potentially fosters cross-strand stabilization and allosteric communication between the filament interior and exterior. We interrupted the potential Interaction by creating either K113E or E195K actin. By combining the two, we also reversed the Interaction with a K113E/E195K (E/K) mutant. In all cases, we isolated viable cells expressing only the mutant actin. Either single mutant cell displays significantly decreased growth in YPD medium. This deficit is rescued in the double mutant. All three mutants display abnormal phalloidin cytoskeletal staining. K113E actin exhibits a critical concentration of polymerization 4 times higher than WT actin, nucleates more poorly, and forms shorter filaments. Restoration of the Ionic bond, E/K, eliminates most of these problems. E195K actin behaves much more like WT actin, indicating accommodation of the neighboring lysines. Both Bni1 and Bnr1 formin FH1-FH2 fragment accelerate polymerization of WT, E/K, and to a lesser extent E195K actin. Bni1p FH1-FH2 dramatically inhibits K113E actin polymerization, consistent with barbed end capping. However, Bnr1p FH1-FH2 restores K113E actin polymerization, forming single filaments. In summary, the proposed Ionic Interaction plays an important role in filament stabilization and in the propagation of allosteric changes affecting formin regulation in an isoform-specific fashion.
-
Importance of a Lys113–Glu195 Intermonomer Ionic Bond in F-actin Stabilization and Regulation by Yeast Formins Bni1p and Bnr1p
Journal of Biological Chemistry, 2013Co-Authors: Melissa Mckane, Peter A. RubensteinAbstract:Abstract Proper actin cytoskeletal function requires actin's ability to generate a stable filament and requires that this reaction be regulated by actin-binding proteins via allosteric effects on the actin. A proposed Ionic Interaction in the actin filament interior between Lys113 of one monomer and Glu195 of a monomer in the apposing strand potentially fosters cross-strand stabilization and allosteric communication between the filament interior and exterior. We interrupted the potential Interaction by creating either K113E or E195K actin. By combining the two, we also reversed the Interaction with a K113E/E195K (E/K) mutant. In all cases, we isolated viable cells expressing only the mutant actin. Either single mutant cell displays significantly decreased growth in YPD medium. This deficit is rescued in the double mutant. All three mutants display abnormal phalloidin cytoskeletal staining. K113E actin exhibits a critical concentration of polymerization 4 times higher than WT actin, nucleates more poorly, and forms shorter filaments. Restoration of the Ionic bond, E/K, eliminates most of these problems. E195K actin behaves much more like WT actin, indicating accommodation of the neighboring lysines. Both Bni1 and Bnr1 formin FH1-FH2 fragment accelerate polymerization of WT, E/K, and to a lesser extent E195K actin. Bni1p FH1-FH2 dramatically inhibits K113E actin polymerization, consistent with barbed end capping. However, Bnr1p FH1-FH2 restores K113E actin polymerization, forming single filaments. In summary, the proposed Ionic Interaction plays an important role in filament stabilization and in the propagation of allosteric changes affecting formin regulation in an isoform-specific fashion.
Maria Rapala-kozik - One of the best experts on this subject based on the ideXlab platform.
-
MECHANISM OF LIGAND-PROTEIN Interaction IN PLANT SEED THIAMINE-BINDING PROTEINS. PRELIMINARY CHEMICAL IDENTIFICATION OF AMINO ACID RESIDUES ESSENTIAL FOR THIAMINE BINDING TO THE BUCKWHEAT-SEED PROTEIN
Biochimie, 1996Co-Authors: Maria Rapala-kozik, Alexander KozikAbstract:Abstract Thiamine-binding protein, isolated from buckwheat seeds, was chemically modified in an attempt to identify amino acid residues involved in protein-thiamine Interaction. No evidence was found in support of specific roles of arginine residues, sulfhydryl groups, amino groups and tyrosine residues. Under carefully controlled reaction conditions (Tris pH 5–6), the modification with I-ethyl-3-(3-dimethylaminopropyl)carbodiimide caused a complete loss of thiamine-binding capacity. Thus, the carboxyl groups seemed to be essential for binding, possibly for Ionic Interaction with protein-bound thiamine cation. A selective modification of histidine residues using diethylpyrocarbonate correlated with a loss of thiamine-binding capacity; the modification and the loss of binding capacity could be reversed with hydroxylamine; some ligand-protection against modification was observed. From Tsou analysis of diethylpyrocarbonate modification and resulting loss of thiamine-binding it was suggested that 1–2 of 20 histidine residues of the protein were essential for thiamine binding. The essential histidine(s) might be present in the binding site and possibly were involved in hydrogen bonding(s) with protein-bound thiamine molecule.
