• Journal of Pharmaceutical Investigation
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• v.19 no.3
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• pp.163-171
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• 1989

To investigate the protein binding characteristics of cephalothin, the effects of ionic strength, pH and temperature on the binding of cephalothin to bovine serum albumin (BSA) were studied by UV difference spectrophotometric method. With increasing ionic strength at constant PH and temperature, association constant decreased, but the number of binding sites sites was about 2 constantly. It may be deduced that the binding process is not only due to electrostatic forces. And the increased association constant at high ionic strength is explained by conformational changes of BSA from complex to subunits. The pH effect on the affinity of interaction indicated that the binding affinity of drug is higher in the neutral region than in the alkaline region. And, at high pH value, the number of binding sites decreased from 2 to 1 because of the conformational changes of BSA in alkaline region. The decrease in binding affinity of BSA to drug with increasing temperature was characteristic of an exothermic reaction. And the negative sign of ${\Delta}G^{\circ}$ meant that the binding process occurs spontaneously under the experimental conditions. In cephalothin-BSA complex formation, since the net enthalpy change value and entropy change value are positive, it is assumed that hydrophobic bindings are predominant in this binding process.

• Journal of Nutrition and Health
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• v.30 no.8
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• pp.930-935
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• 1997

Fatty acid binging proteins(FABP) are distinct but related gene productes which are found in many mamalian cell types. FABP bind long chain fatty acids in vitro. However, their functions and mechanisms of action, in vivo, remain unknown . Also not known is whether all FABP function similaryly in their respective cell types. or whether different FABP have unique functions. The puropose of the present study was to assess whether different members of the FABP family exhibit different structural and function properties. A comparison was made between heart(H-FABP) and liver (L-FABP). The results show that the binding sites of both FABP are hydrophobic in nature, although the L-FABP site is more nonpolar than the H-FABP site. Additionally, the bound ligand experiences less motional constraint within the H-FABP binding site than within the L-FABP binding site. In accordance with these differences in structural properties, it was found that anthroyloxy-fatty acid transfer from H-FABP to membranes is markedly faster than from L-FABP. moreover, the mechanism of fatty acid transfer to phospholipid membranes appears to occur via transient collisional interactions between H-FABP and membranes. In contrast , transfer of fatty acid from L-FABP occurs via an aqueous diffusion mechanism.

• Journal of Microbiology
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• v.37 no.1
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• pp.21-26
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• 1999

Two novel calcium-binding proteins, named CAB-I and CAB-II, have been isolated from Streptomyces coelicolor. Purification of the calcium-binding proteins involved heat treatment, fractionation with ammonium sulfate, acid treatment, anion exchange and hydrophobic interaction column chromatography, FPLC gel filtration, and preparative isoelectric focusing. A chelex competitive assay and 45Ca autoradiography verified the calcium-binding ability of the proteins. The major band CAB-II has an apparent molecular weight of 26,000 determined by SDS-polyacrylamide gel electrophoresis and 340,000 determined by gel filtration. The isoelectric point of this molecule showed the acidic nature of the molecule. N-terminal amino acid sequence analysis shows homology to rat Ca2+/calmodulin-dependent protein kinase-II (CAB-II) and yeast phosphoprotein phosphatase (CAB-I).

• Molecules and Cells
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• v.39 no.3
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• pp.217-228
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• 2016

To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their antiproliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity ($IC_{50}$: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity ($K_D$: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCIN82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5.

• Bulletin of the Korean Chemical Society
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• v.31 no.2
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• pp.442-446
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• 2010

Adenosine 3',5'-cyclic monophosphate (cAMP) is a second messenger responsible for a multitude of cellular responses. In this study, we utilized $\beta$-cyclodextrin ($\beta$-CD) as an artificial receptor with a hydrophobic cavity to elucidate the inclusion kinetics of cAMP in a hydrophobic environment using the ultrasonic relaxation method. The results revealed that the interaction of cAMP with $\beta$-CD followed a single relaxation curve as a result of host-guest interactions. The inclusion of cAMP into the $\beta$-CD cavity was found to be a diffusion-controlled reaction. The dissociation of cAMP from the $\beta$-CD cavity was slower than that of adenosine 5'-monophosphate (AMP). The syn and anti glycosyl conformations of adenine nucleotides are considered to play an important role in formation of the inclusion complex. Taken together, our findings indicate that hydrophobic interactions are involved in the inclusion complex formation of cAMP with $\beta$-CD and provide insight into the interactions of cAMP with cAMP-binding proteins.

• Journal of the Korean Magnetic Resonance Society
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• v.15 no.1
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• pp.1-13
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• 2011

9Pbw2 is a 9-mer analog of protaetiamycine derived from the larvae of the beetle Protaetia brevitarsis. Previously, we designed four 9-mer peptide analogues to optimize the balance between the hydrophobicity and cationicity of the peptides and to increase bacterial cell selectivity. Among them, 9Pbw2 has high antibacterial activity without cytotoxicity. The results obtained in previous study suggest that the bactericidal action of 9Pbw2 may be attributed to the inhibition of the functions of intracellular components after penetration of the bacterial cell membrane. In order to understand structure-activity relationships, we determined the three-dimensional structure of 9Pbw2 in 200 mM DPC micelle by NMR spectroscopy. 9Pbw2 has one hydrophobic turn helix from $Trp^3$ to $Arg^8$ and positively charged residues at the N- and C-terminus. This result suggested that positively charged residues from position at the C-terminus in 9Pbw2 may be important for the primary binding to the negatively charged phospholipid head groups in bacterial cell membranes and hydrophobic residues in the middle portion face toward the acyl chains of the hydrophobic lipid in the bacterial cell membrane.

• Bulletin of the Korean Chemical Society
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• v.35 no.3
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• pp.783-792
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• 2014

The binding interaction between a hemolytic peptide ${\delta}$-lysin and a zwitterionic lipid bilayer POPC was investigated through a series of molecular dynamics (MD) simulations. ${\delta}$-Lysin is a 26-residue, amphipathic, ${\alpha}$-helical peptide toxin secreted by Staphylococcus aureus. Unlike typical antimicrobial peptides, ${\delta}$-lysin has no net charge and it is often found in aggregated forms in solution even at low concentration. Our study showed that only the monomer, not dimer, inserts into the bilayer interior. The monomer is preferentially attracted toward the membrane with its hydrophilic side facing the bilayer surface. However, peptide insertion requires the opposite orientation where the hydrophobic side of peptide points toward the membrane interior. Such orientation allows the charged residues, Lys and Asp, to have stable salt bridges with the lipid head-group while the hydrophobic residues are buried deeper in the hydrophobic lipid interior. Our simulations suggest that breaking these salt bridges is the key step for the monomer to be fully inserted into the center of lipid bilayer and, possibly, to translocate across the membrane.

• Ocean and Polar Research
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• v.33 no.2
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• pp.159-169
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• 2011

Antifreeze proteins (AFPs) have ice binding affinity, depress freezing temperature and inhibit ice recystallization which protect cellular membranes in polar organisms. Recent structural studies of antifreeze proteins have significantly expanded our understanding of the structure-function relationship and ice crystal growth inhibition. Although AFPs (Type I-IV AFP from fish, insect AFP and Plant AFP) have completely different fold and no sequence homology, they share a common feature of their surface area for ice binding property. The conserved ice-binding sites are relatively flat and hydrophobic. For example, Type I AFP has an amphipathic, single ${\alpha}$-helix and has regularly spaced Thr-Ala residues which make direct interaction with oxygen atoms of ice crystals. Unlike Type I AFP, Type II and III AFP are compact globular proteins that contain a flat ice-binding patch on the surface. Type II and Type III AFP show a remarkable structural similarity with the sugar binding lectin protein and C-terminal domain of sialic acid synthase, respectively. Type IV is assumed to form a four-helix bundle which has sequence similarity with apolipoprotein. The results of our modeling suggest an ice-binding induced structural change of Type IV AFP. Insect AFP has ${\beta}$-helical structure with a regular array of Thr-X-Thr motif. Threonine residues of each Thr-X-Thr motif fit well into the ice crystal lattice and provide a good surface-surface complementarity. This review focuses on the structural characteristics and details of the ice-binding mechanism of antifreeze proteins.

• BMB Reports
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• v.32 no.6
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• pp.594-598
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• 1999

The Jun and Fos families of eukaryotic transcription factors form heterodimers capable of binding to their cognate DNA enhancer elements. We are interested in searching for inhibitors or antagonists of the binding of the Jun-Fos heterodimer to the activator protein-1 (AP-1) site. The basic-region leucine zipper (bZIP) domain of c-Fos was expressed as a fusion protein with glutathione S-transferase, and allowed to form a heterodimer with the bZIP domain of c-Jun. The heterodimer was bound to glutathione-agarose, to which were added radiolabeled AP-1 nucleotides. After thorough washing, the gel-bound radioactivity was counted. The assay is faster than the coventional electrophoretic mobility shift assay because the gel electrophoresis step and the autoradiography step are eliminated. Moreover, the assay is very sensitive, allowing the detection of picomolar quantities of nucleotides, and is not affected by up to 50% dimethylsulfoxide, a solvent for hydrophobic inhibitors. Curcumin and dihydroguaiaretic acid, recently known inhibitors of Jun-Fos-DNA complex formation, were applied to this Jun-GST-fused Fos system and revealed to decrease the dimer-DNA binding.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.3
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• pp.385-393
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• 1998

Human neutrophil elastase (HNElastase, EC 3.4.21.37), a causative factor of inflammatory diseases, was purified by Ultrogel AcA54 gel filtration and CM-Sephadex ion exchange chromatography. HNElastase was inhibited by phenylbutazone in a concentration dependent manner up to 0.4 mM, but as the concentration increased, the inhibitory effect gradually diminished. Binding of phenylbutazone to the human neutrophil elastase caused strong Raman shifts at 200, 440, and 1194 $cm^{-1}$. The peak at 1194 $cm^{-1}$ might be evidence of the presence $of\;-N=N-{\Phi}$ radical. The core area of the elastase, according to the visual molecular model of human neutrophil elastase, was structurally stable. A deeply situated active center was at the core area surrounded by hydrophobic amino acids. Directly neighboring the active site was one positively charged atom and two atoms carrying a negative charge, which enabled the enzyme and the drug to form a strong interaction. Phenylbutazone may form a binding, similar to a key & lock system to the atoms carrying opposite charges near the active site of the enzyme molecule. Furthermore, the hydrophobicity of the surrounding amino acid near the active site seemed to enhance the binding strength of phenylbutazone. Binding of phenylbutazone near the active site may cause masking of the active site, preventing the substrate from approaching the active site and inhibiting elastase activity.