• Journal of Life Science
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• v.27 no.12
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• pp.1410-1414
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• 2017

Escherichia coli tryptophan synthase (TS) contains ${\alpha}_2{\beta}_2$, which catalyzes the final two steps in Trp biosynthesis. A molecular tunnel exists between the two active sites of ${\alpha}$ and ${\beta}$ subunits in TS. Via intersubunit communication, TS increases catalytic efficiency, including substrate channeling. The ${\beta}$ subunit of TS is composed of two domains, one of which, the COMM (communication) domain, plays an important role in intersubunit communication. The ${\alpha}$ subunit has a TIM barrel structure. This protein has functional regions at the C terminal of ${\beta}$ pleated sheets and in its loop regions. Three regions of the ${\alpha}$ subunit (${\alpha}L6$ [${\alpha}-loop$ L6], ${\alpha}L2$, and ${\alpha}L3$) are implicated in intersubunit communication. In the present study, conformational changes in ${\alpha}L6$ were monitored by measuring the sensitivity of mutant proteins in these regions to trypsin. The addition of a ${\alpha}$ subunit-specific ligand, D,L-${\alpha}$-glycerophosphate (GP), partially restored the sensitivity of mutant proteins to trypsin. In contrast, the addition of the ${\beta}$ subunit-specific ligand L-serine (Ser) resulted in varied sensitivity to trypsin, with an increase in PT53 (substitution of Pro with Thr at residue 53) and DG56, decrease in NS104 and wild type, and no change in GD51 and PH53. This finding may be related to several reaction intermediates formed under this condition. The addition of both GP and Ser led to a highly stable state of the complex. The present results are consistent with the current model. The method used herein may be useful for screening residues involved in intersubunit communication.

• Molecules and Cells
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• v.37 no.3
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• pp.264-269
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• 2014

The molecular interaction between tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins plays an essential role in the transcription-independent apoptotic pathway of p53. In this study, we investigated the binding of p53 DNA-binding domain (p53DBD) with the anti-apoptotic Bcl-2 family proteins, Bcl-w, Mcl-1, and Bcl-2, using GST pull-down assay and NMR spectroscopy. The GST pull-down assays and NMR experiments demonstrated the direct binding of the p53DBD with Bcl-w, Mcl-1, and Bcl-2. Further, NMR chemical shift perturbation data showed that Bcl-w and Mcl-1 bind to the positively charged DNA-binding surface of p53DBD. Noticeably, the refined structural models of the complexes between p53DBD and Bcl-w, Mcl-1, and Bcl-2 showed that the binding mode of p53DBD is highly conserved among the anti-apoptotic Bcl-2 family proteins. Furthermore, the chemical shift perturbations on Bcl-w, Mcl-1, and Bcl-2 induced by p53DBD binding occurred not only at the p53DBD-binding acidic region but also at the BH3 peptide-binding pocket, which suggests an allosteric conformational change similar to that observed in Bcl-$X_L$. Taken altogether, our results revealed a structural basis for a conserved binding mechanism between p53DBD and the anti-apoptotic Bcl-2 family proteins, which shed light on to the molecular understanding of the transcription-independent apoptosis pathway of p53.

• Korean Journal of Microbiology
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• v.47 no.3
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• pp.200-208
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• 2011

Most problematic antibiotic resistance mechanism for MLS (macrolide-lincosamide-streptogramn B) antibiotics encountered in clinical practice is mono- or dimethylation of specific adenine residue at 2058 (E. coli coordinate) of 23S rRNA which is performed by Erm (erythromycin ribosome resistance) protein through which bacterial ribosomes reduce the affinity to the antibiotics and become resistant to them. ErmSF is one of the four gene products produced by Streptomyces fradiae to be resistant to its own antibiotic, tylosin. Unlike other Erm proteins, ErmSF harbors idiosyncratic long N-terminal end region (NTER) 25% of which is comprised of arginine well known to interact with RNA. Furthermore, NTER was found to be important because when it was truncated, most of the enzyme activity was lost. Based on these facts, capability of NTER peptide to inhibit the enzymatic activity of ErmSF was sought. For this, expression system for two different proteins to be expressed in one cell was developed. In this system, two plasmids, pET23b and pACYC184 have unique replication origins to be compatible with each other in a cell. And expression system harboring promoter, ribosome binding site and transcription termination signal is identical but disparate amount of protein could be expressed according to the copy number of each vector, 15 for pACYC and 40 for pET23b. Expression of NTER peptide in pET23b together with ErmSF in pACYC 184 in E. coli successfully gave more amounts of NTER than ErmSF but no inhibitory effects were observed suggesting that there should be dynamicity in interaction between ErmSF and rRNA rather than simple and fixed binding to each other in methylation of 23S rRNA by ErmSF.

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.278-285
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• 2016

The bifunctional PheA protein, having chorismate mutase and prephenate dehydratase (CMPD) activities, is one of the key regulatory enzymes in the aromatic amino acid biosynthesis in Escherichia coli, and is negatively regulated by an end-product, phenyalanine. Therefore, PheA protein has been thought as useful for protein engineering to utilize mass production of essential amino acid phenylalanine. To obtain feedback resistant PheA protein against phenylalanine, we mutated by using random mutagenesis, extensively screened, and obtained $pheA^{FBR}$ gene encoding a feedback resistant PheA protein. The mutant PheA protein contains substitution of Leu to Phe at the position of 118, displaying that higher affinity (about $290{\mu}M$) for prephenate in comparison with that (about $850{\mu}M$) of wild type PheA protein. Kinetic analysis showed that the saturation curve of $PheA^{FBR}$ against phenyalanine is hyperbolic rather than that of $PheA^{WT}$, which is sigmoidal, indicating that the L118F mutant enzyme has no cooperative effects in prephenate binding in the presence of phenylalanine. In vitro enzymatic assay showed that the mutant protein exhibited increased activity by above 3.5 folds compared to the wild type enzyme. Moreover, L118F mutant protein appeared insensitive to feedback inhibition with keeping 40% of enzymatic activity even in the presence of 10 mM phenylalanine at which the activity of wild type $PheA^{WT}$ was not observed. The substitution of Leu to Phe in CMPD may induce significant conformational change for this enzyme to acquire feedback resistance to end-product of the pathway by modulating kinetic properties.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.18 no.1
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• pp.99-132
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• 1992

Polypeptide has been used broadly as an active ingredient in cosmetics We thought it is very important to investigate the adsorption behavior of polypeptide in order to pre-estimate the effect of these polypeptides. For the study of polypeptide adsorption, we have investigated complex formation of basic homopolypeptides, poly(L-proline) Form I [PLP(I)], Form II [PLP(II)] and poly(4-hydroxy-L-proline) (PHLP) with acidic homopolypeptides, poly(L-glutamic acid) (PLGA), poly(D-glutamic acid) (PDGA) and poly(L-aspartic acid) (PLAA) through hydrogen bonding in a hydroalcoholic medium with viscometer, 1ight scatter, pH meter and circular dicroism (CD). The polypeptides used in this study have helical structure in some conditions. The result exhibited that al 1 the complexes were formed as the composition of basic/acidic homopolypeptide : L:2 irrespective of the complex systems used. A more favorable complex is formed in the PLP(II)-PLGA system than PHLP-PLGA because PLP(II) has a more flexible helical conformation, whereas PHLP has a more rigid helical conformation. The right-handed helix PLGA formed the complex favorably and quickly with the left-handed helix PLP(II), whereas the left-handed helix PDGA formed the complex favorably with the right-handed helix PLP(I). The effect of side chain of the acidic homopolypeptides on the complexation was also studied. The result showed that more favorable condition for the complexation was PLGA-PLP(II) system which has longer side chain at acidic homopolypeptide than PLAA - PLP(II). All the above facts were well supported by CD measurement for the complex systems. By the CD spectra for the complexes we could deduce the conformational change of each homopolypeptide in the complexes On the basis of the above results, we performed the adsorption test of PLP(I, II) and PHLP on the hair having a left-handed helix. The adsorption amount of each polypeptide was analyzed by HPLC. The result showed that PLP(I) was adsorbed more than PLP(II), PLP(II) was adsorbed more than PHLP on the hair. On adsorbing polypeptides having a helical structure on the hair through hydrogen bonding, it could be concluded that the helical polypeptides having the opposite directional structure to the hair are adsorbed more than those having the same directional structure with the hair and also the polypeptides having a flexible conformation are adsorbed more than those having a rigid conformation.

• Economic and Environmental Geology
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• v.44 no.6
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• pp.485-492
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• 2011

Sorption of cyanide on granular activated carbon and attenuation of the cyanide by UV-light over a wide range of conditions such as pH and concentration were investigated through batch experiments. Cyanide uptake by activated carbon is much effective at $[CN]_{ini}$ < 2 mg/L. The sorption of cyanide on activated carbon at pH 7.0 is greater than that of pH 9.0. It is found that the ratio of CN uptake to CN in solution increases at pH 9.0 whereas at pH 7.0 the ratio decreases, suggesting that reactivity of activated carbon increases as a function of pH. The sorption of cyanide rapidly increases during the first 30 min, followed by sharp desorption until 3hr, and then the sorption increases and reaches the maximum sorption during the duration of experiments, implying that the sorption mode could be changed through conformational change during the initial stage of the cyanide uptake by activated carbon. Total amount of cyanide desorbed from the activated carbon during the period of desorption experiments is less than 1.5% of total sorbed cyanide, indicative of strong and stable sorption of cyanide on the activated carbon. The sorption with mixture of activated carbon and Ham-Baek sludge shows less effective on the removal of the cyanide. It is noted that UV-light is much effective on the removal of cyanide but also the attenuation is achieved until $[CN]_{tot}$ is up to 10 mg/L. Our findings demonstrate that both activated carbon and UV-light are very effective on the attenuation of cyanide over a wide range of environmental conditions.