ParABS, an important DNA partitioning process in chromosome segregation, includes ParA (an ATPase), ParB (a
par
S binding protein) and
parS
(a centromere-like DNA). The homologous proteins of ParA and ParB in
Helicobacter pylori
are
Hp
Soj and
Hp
Spo0J, respectively. We analyzed the ATPase activity of
Hp
Soj and found that it is enhanced by both DNA and
Hp
Spo0J. Crystal structures of
Hp
Soj and its DNA complexes revealed a typical ATPase fold and that it is dimeric. DNA binding by
Hp
Soj is promoted by ATP. The
Hp
Soj–ATP–DNA complex non-specifically binds DNA through a continuous basic binding patch formed by lysine residues, with a single DNA-binding site. This complex exhibits a DNA-binding adept state with an active ATP-bound conformation, whereas the
Hp
Soj–ADP–DNA complex may represent a transient DNA-bound state. Based on structural comparisons,
Hp
Soj exhibits a similar DNA binding surface to the bacterial ParA superfamily, but the archaeal ParA superfamily exhibits distinct non-specific DNA-binding via two DNA-binding sites. We detected the
Hp
Spo0J–
Hp
Soj–DNA complex by electron microscopy and show that this nucleoid-adaptor complex (NAC) is formed through
Hp
Soj and
Hp
Spo0J interaction and
parS
DNA binding. NAC formation is promoted by
Hp
Soj participation and specific
parS
DNA facilitation.
Heparan sulfate (HS) is a linear sulfated polysaccharide that mediates protein activities at the cell-extracellular interface. Its interactions with proteins depend on the complex patterns of sulfonations and sugar residues. Previously, we synthesized all 48 potential disaccharides found in HS and used them for affinity screening and X-ray structural analysis with fibroblast growth factor-1 (FGF1). Herein, we evaluated the affinities of the same sugars against FGF2 and determined the crystal structures of FGF2 in complex with three disaccharides carrying N-sulfonated glucosamine and 2-O-sulfonated iduronic acid as basic backbones. The crystal structures show that water molecules mediate different interactions between the 3-O-sulfonate group and Lys125. Moreover, the 6-O-sulfonate group forms intermolecular interactions with another FGF2 unit apart from the main binding site. These findings suggest that the water-mediated interactions and the intermolecular interactions influence the binding affinity of different disaccharides with FGF2, correlating with their respective dissociation constants in solution.
The influence of three sugar osmolytes on the refolding of guanidine hydrochloride- (GdnHCl-) denatured trehalose-6-phosphate hydrolase of Bacillus licheniformis (BlTreA) was studied by circular dichroism (CD) spectra, fluorescence emission spectra, and the recovery of enzymatic activity. These experimental results clearly indicated that sorbitol, sucrose, and trehalose at a concentration of 0.75 M improved the refolding yields of GdnHCl-denatured BlTreA, probably due to the fact that these sugars favored the formation of tertiary architectures. Far-UV CD measurements demonstrated the ability of sugar osmolytes to shift the secondary structure of GdnHCl-denatured enzyme towards near-native conformations. ANS fluorescence intensity measurements revealed a reduction of exposed hydrophobic surfaces upon the treatment of denatured enzyme with sugar osmolytes. These observations suggest that sugar osmolytes possibly play a chaperone role in the refolding of chemically denatured BlTreA.
Abstract:In this study, we synthesized water-soluble hyperbranched poly(amido acid)s (HBPAAs) featuring multiple terminal CO 2 H units and internal tertiary amino and amido moieties and then used them in conjunction with an in situ Fe 2+/ Fe 3+ co-precipitation process to prepare organic/magnetic nanocarriers comprising uniformly small magnetic iron oxide nanoparticles (NP) incorporated within the globular HBPAAs. Transmission electron microscopy revealed that the HBPAA-γ-Fe 2 O 3 NPs had dimensions of 6-11 nm, significantly smaller than those of the pristine γ-Fe 2 O 3 (20-30 nm). Subsequently, we covalently immobilized a bacterial γ-glutamyltranspeptidase (BlGGT) upon the HBPAA-γ-Fe 2 O 3 nanocarriers through the formation of amide linkages in the presence of a coupling agent. Magnetization curves of the HBPAA-γ-Fe 2 O 3 /BlGGT composites measured at 300 K suggested superparamagnetic characteristics, with a saturation magnetization of 52 emu g support; this sample provided a 48% recovery of the initial activity. The immobilized enzyme could be recycled 10 times with 32% retention of the initial activity; it had stability comparable with that of the free enzyme during a storage period of 63 days. The covalent immobilization and stability of the enzyme and the magnetization provided by the HBPAA-γ-Fe 2 O 3 NPs suggests that this approach could be an economical means of depositing bioactive enzymes upon nanocarriers for BlGGT-mediated bio-catalysis.
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