Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulin-like seven-stranded anti-parallel ...β-barrel. Because of the labile chromophore reflecting its extremely strong DNA cleavage activity and complete stabilization in the complex, holo-NCS has attracted much attention in clinical use as well as for drug delivery systems. Despite many structural analyses for holo-NCS, the chromophore-releasing mechanism to trigger prompt attacks on the target DNA is still unclear. We determined the three-dimensional structure of the protein and the internal motion by multinuclear NMR to investigate the releasing mechanism. The internal motion studied by 13C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the β-barrel in aqueous solution. This agrees with the refined NMR solution structure, which has good convergence in the loop regions. We also showed that the chromophore displayed a similar internal motion as the protein moiety. The structural comparison between the refined solution structure and x-ray crystal structure indicated characteristic differences. Based on the findings, we proposed the chromophore-releasing mechanism by a three-state equilibrium, which sufficiently describes both the strong binding and the prompt releasing of the chromophore. We demonstrated that we could bridge the dynamic properties and the static structure features with simple kinetic assumptions to solve the biochemical function.
Staphylococcal protein A is a cell wall constituent of most strains of
Staphylococcus aureus, and it is characterized by its binding affinity to some immunological classes. A mutated low molecular ...weight type protein A (LPA; Mwt=27
kDa) which consists of the domains, E, D, A, B and 13 residues of the C-domain was prepared in this study. Since LPA does not possess a cell wall-bound region in contrast to wild-type protein A (WPA; Mwt=42
kDa), we have established a methodology of large scale purification of LPA without using any extracellular expression systems such as
Escherichia coli. Using this relatively abundant protein, the immobilization of the LPA with silk fibroin of
Bombyx mori was performed. Thermal stability of LPA immobilized with silk fibroin is higher than that of free LPA at high temperature judging from the immunoglobulin G (IgG)-binding affinity. However, the apparent value of its affinity decreased relative to that of immobilized WPA. These results indicate that structural information is essential to explore improvement of IgG-binding affinity of immobilized LPA. Therefore, secondary structure of free LPA was detected by its characteristic helical pattern in circular dichroism (CD) in aqueous solution. In addition to this, tertiary fold of four IgG-binding domains were investigated by two-dimensional
1H-NMR spectra. Four significantly high-field shifted cross-peaks attributed to methyl signals of alanine residues suggest that all four domains pack into a three helix bundle motif in solution. These structural data and properties of IgG-binding affinity suggest that spatial arrangement of four IgG-binding domains are packed into a compact globular molecular shape. This causes a certain active site of immobilized LPA to be buried in the silk fibroin fiber.
We found two types of novel alkaline metalloendopeptidases (AP1 and AP2) from a marine bacterium, isolated from the intestine of a five-barred goatfish (Parupeneus trifasciatus) and identified as ...Vibrio sp. (NUF-BPP1). We studied the structure-function relationship of these marine bacterial proteases. The electrophoretically homogeneous proteases had a molecular mass of 48 kDa for AP1 and 36 kDa for AP2 on SDS-PAGE, and showed optimum activity at around pH 9.5-10.0 (casein as substrate). Calcium chloride (5 mm) stabilized the activities over pH 6-11, but o-phenanthroline and EDTA inhibited the activities of both AP1 and AP2. The EDTA-inactivated proteases were partly restored to activity by addition of either zinc or calcium. Sodium chloride (3.5 m) increased the activities toward Z-Gly-Leu-NH
2
. N-Terminal sites of hydrophobic amino acid residues (Leu, Ile, Phe, Tyr, and Trp) of the peptide substrates were cleaved by AP1 and by AP2. Autolysis of AP1 in the absence of calcium ion probably produced AP2 by releasing a fragment (molecular mass of about 12kDa) from the C-terminal end of AP1.
The planar structure of a new polyene macrolide antibiotic, YS-822A, which was isolated from the culture filtrate of a mutant strain H-8 of Streptoverticillium eurocidicum var. asterocidicus S-822, ...was established as I on the basis of spectroscopic evidences and by comparison of spectroscopic data with nystatin A1 (II) and amphotericin A (III), representative polyene macrolide antibiotics. Molecular formula of YS-822A was established as C37H59NO14 (MW 741) by elemental analysis, NMR, and FAB mass spectra. The UV spectrum of YS-822A was very similar to that of nystatin A1, suggesting that YS-822A also has a conjugated all-trans-tetraene moiety. 1H and 13C NMR spectra of YS-822A showed a number of broad and overlapped signals, but the 1H-1H and 13C-1H COSY spectra implied the existence of a mycosamine moiety and several other partial structures. The connectivity of these partial structures was established by extensive 2D NMR experiments, including homonuclear Hartmann-Hahn and heteronuclear multiple-bond connectivity measurements, which led to the determination of the gross planar structure of YS-822A as I.
A new polyene macrolide antibiotic, YS-822A was isolated from the culture filtrate of a mutant strain H-8 of Streptoverticillium eurocidicum var. asterocidicus S-822. Whereas the original S-822 ...strain produced not only YS-822 substances but also teleocidin as by-product which is well-known as a strong carcinogenic promotor, the mutagenized H-8 strain produced the antibiotic with only a trace amount of teleocidin. Chemical and biological characterizations of the antibiotic revealed that YS-822A (molecular formula: C37H59NO14) is a new polyene macrolide with a wide antifungal spectrum and a low acute toxicity.
We found two types of novel alkaline metalloendopeptidases (AP1 and AP2) from a marine bacterium, isolated from the intestine of a five-barred goatfish (Parupeneus trifasciatus) and identified as ...Vibrio sp. (NUF-BPP1). We studied the structure-function relationship of these marine bacterial proteases. The electrophoretically homogeneous proteases had a molecular mass of 48 kDa for AP1 and 36 kDa for AP2 on SDS-PAGE, and showed optimum activity at around pH 9.5-10.0 (casein as substrate). Calcium chloride (5 mM) stabilized the activities over pH 6-11, but o-phenanthroline and EDTA inhibited the activities of both AP1 and AP2. The EDTA-inactivated proteases were partly restored to activity by addition of either zinc or calcium. Sodium chloride (3.5M) increased the activities toward Z-Gly-Leu-NH2. N-Terminal sites of hydrophobic amino acid residues (Leu, Ile, Phe, Tyr, and Trp) of the peptide substrates were cleaved by AP1 and by AP2. Autolysis of AP1 in the absence of calcium ion probably produced AP2 by releasing a fragment (molecular mass of about 12kDa) from the C-terminal end of AP1.
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