• BMB Reports
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• 제32권6호
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• pp.573-578
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• 1999

Treatment of Hafnia alvei aspartase with limited tryptic digestion resulted in a marked increase in enzymatic activity. The activation required a few minutes to attain maximum level and, thereafter, the activity gradually decreased to complete inactivation. The degree of cleavage associated with the activation was extremely small as judged by SDS-PAGE. Upon activation, the optimum pH and temperature were essentially unchanged. When trypsin-activated enzyme was denatured in 4 M guanidine-HCI followed by removal of the denaturant by dilution, the restoration of activity was similar (40%) to that of the native enzyme, indicating a degree of stability. The $pK_a$ obtained on the acidic side and the $pK_b$ obtained on the basic side of trypsin-activated aspartase were 6.6 and 8.6, respectively, the same as those of the native aspartase, indicating that aspartase may exist in a stable conformation after limited tryptic digestion. These results indicate that the activation of H. alvei may be mediated by a conformational change away from the active site of individual subunits.

• BMB Reports
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• 제39권3호
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• pp.319-328
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• 2006

SecA protein, a cytoplasmic ATPase, plays a central role in the secretion of signal peptide-containing proteins. Here, we examined effects of signal peptide and ATP on the oligomerization, conformational change, and membrane binding of SecA. The wild-type (WT) signal peptide from the ribose-binding protein inhibited ATP binding to soluble SecA and stimulated release of ATP already bound to the protein. The signal peptide enhanced the oligomerization of soluble SecA, while ATP induced dissociation of SecA oligomer. Analysis of SecA unfolding with urea or heat revealed that the WT signal peptide induces an open conformation of soluble SecA, while ATP increased the compactness of SecA. We further obtained evidences that the signal peptide-induced oligomerization and the formation of open structure enhance the membrane binding of SecA, whereas ATP inhibits the interaction of soluble SecA with membranes. On the other hand, the complex of membrane-bound SecA and signal peptide was shown to resume nucleotide-binding activity. From these results, we propose that the translocation components affect the degree of oligomerization of soluble SecA, thereby modulating the membrane binding of SecA in early translocation pathway. A possible sequential interaction of SecA with signal peptide, ATP, and cytoplasmic membrane is discussed.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1582-1589
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• 2009

Bacterial UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first step of bacterial cell wall synthesis. We identified thimerosal, thiram, and ebselen as effective inhibitors of Haemophilus influenzae MurA by screening a chemical library that consisted of a wide range of bioactive compounds. When MurA was preincubated with these inhibitors, their 50% inhibitory concentrations ($IC_{50}s$) were found to range from 0.1 to $0.7\;{\mu}M$. In particular, thimerosal suppressed the growth of several different Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium at a concentration range of $1-2\;{\mu}g/ml$. These inhibitors covalently modified the cysteine residue near the active site of MurA. This modification changed the open conformation of MurA to a more closed configuration, which may have prevented the necessary conformational change from occurring during the enzyme reaction.

• Journal of Microbiology and Biotechnology
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• 제28권9호
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• pp.1457-1466
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• 2018

In the present study, the stabilizing effect of four different biological osmolytes on Bacillus licheniformis ${\gamma}$-glutamyl transpeptidase (BlGGT) was investigated. BlGGT appeared to be stable under temperatures below $40^{\circ}C$, but the enzyme retained less than 10% of its activity at $60^{\circ}C$. The tested osmolytes exhibited different degrees of effectiveness against temperature inactivation of BlGGT, and sucrose was found to be the most effective among these. The use of circular dichroism spectroscopy for studying the secondary structure of BlGGT revealed that the temperature-induced conformational change of the protein molecule could be prevented by the osmolytes. Consistently, the molecular structure of the enzyme was essentially conserved by the osmolytes at elevated temperatures as monitored by fluorescence spectroscopy. Sucrose was further observed to counteract guanidine hydrochloride (GdnHCl)-and urea-induced denaturation of BlGGT. Taken together, we observed evidently that some well-known biological osmolytes, especially sucrose, make a dominant contribution to the structural stabilization of BlGTT.

• 한국자기공명학회논문지
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• 제15권2호
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• pp.175-186
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• 2011

Amyloid fibrils have long been known to be the well known ${\alpha}$-helix to ${\beta}$-sheet transition characterizing the conversion of cellular to scrapie forms of the prion protein. A very short sequence of Yeast prion-like protein, GNNQQNY (SupN), is responsible for aggregation that induces diseases. KSI-fused tandem repeats of SupN vector are constructed and used to express SupN peptide in Escherichia coli (E.Coli). A method for a production, purification, and cleavage of tandem repeats of recombinant isotopically enriched SupN in E. coli is described. This method yields as much as 20 mg/L of isotope-enriched fusion proteins in minimal media. Synthetic SupN peptides and $^{13}C$ Gly labeled SupN peptides are studied by Congo Red staining, Birefringence and transmission electron microscopy to characterize amyloid fibril formation. To get a better understanding of aggregation-structure relationship of 7 residues of Yeast prion-like protein, the change of a conformational structure will be studied by $^{13}C$ solid-state nmr spectroscopy as powder of both amorphous and fibrillar forms.

• Asian Pacific Journal of Cancer Prevention
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• 제13권4호
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• pp.1599-1603
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• 2012

Objectives: Since methylenetetrahydrofolate reductase (MTHFR) maintains the balance of circulating folate and methionine and blocks the formation of homocysteine, its regulation in relation to different cancers has extensively been studied in different populations. However, information on Pakistani breast cancer patients is lacking. The MTHFR gene has two most common mutations that are single nucleotide additions which result in change of amino acids C677T to Ala222val and A1298C to Glu429Ala. Methodology: 110 sporadic breast patients with no prior family history of cancer or any other type of genetic disorders along with 110 normal individuals were screened for mutations in exons 1 to exon 9 using single strand conformational polymorphism, RFLP and sequencing analyzer. Results: The p values for the 677CC, 677CT, and 677TT genotypes were 0.223, 0.006, and 0.077, respectively. Those for the 1298AA, 1298AC, and 1298CC genotypes were 0.555, 0.009, and 0.003, respectively. Conclusions: We found an overall a significant, weak inverse association between breast cancer risk and the 677TT genotype and an inverse association with the 1298C variant. These results for MTHFR polymorphism might be population specific in sporadic breast cancer affected patients but many other factors need to be excluded before making final conclusions including folate intake, population and disease heterogeneity.

• Applied Microscopy
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• 제17권1호
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• pp.29-46
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• 1987

To study hexavalent chromium effects on mitochondrial electron transport, the activities of electron transport enzymes and conformational change of mitochondria treated with $40{\mu}M$ of sodium dichromate ($Na_{2}Cr_{2}O_{7}\;2H_{2}O$) were investigated. And so were those of liver mitochondria isolated from mouse intraperitoneally injected with sodium dichromate, 40mg per kg body weight. On both treatment with chromium(VI), the activities of electron transfer enzymes (Complex I and IV) were increased to some extent and the ultrastructural transformation of mitochondria from a condensed to an orthodox conformation was inhibited under State IV respiration. These results represent' inhibitory effect of hexavalent chromium on electron transport without inhibiting electron transfer enzymes (Complex I and IV) in mitochondria. On intraperitoneal treatment with hexavalent chromium as sodium dichromate and trivalent chromium as chromic chloride, containing 37.5 mg of chromium per kg body weight, respectively, the activities of electron transfer enzymes of liver isolated from mouse with chromium(VI) was reduced, but that with chromium(III) was not affected. And with chromium(VI), all mice after 12 hours of treatment died, only after 6 hours survived. With chromium(III), however, all survived. This indicates that hexavalent chromium is more toxic than trivalent chromiumin mouse liver.

• Journal of Photoscience
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• 제12권3호
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• pp.175-183
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• 2005

Plants dissipate excess excitation energy from their photosynthetic apparatus by a process called non-photochemical quenching (NPQ). The major part of NPQ is energy dependent quenching (qE) which is dependent on the thylakoid pH and regulated by xanthophyll cycle carotenoids associated with photosystem (PS) II of higher plants. The acidification of the lumen leads to protonation and thus conformational change of light harvesting complex (LHC) proteins as well as PsbS protein of PSII, which results in the induction of qE. Although physiological importance of qE has been well established, the mechanistic understanding is rather insufficient. However, recent finding of crystal structure of LHCII trimer and identification of qE mutants in higher plants and algae enrich and sharpen our understanding of this process. This review summarizes our current knowledge on the qE mechanism. The nature of quenching sites and components involved in this process, and their contribution and interaction for the generation of qE appeared in the proposed models for the qE mechanism are discussed.

• 식품기술
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• 제17권4호
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• pp.98-106
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• 2004

Two mutant enzymes of $\gamma$-glutamylcysteine synthetase ($\gamma$-GCS) which catalyzed the synthesis of $\gamma$-glutamylcysteine from L-glutamic acid and L-cysteine in the presence of ATP, were prepared bypoint mutation of $\gamma$-GCS gene with site-directed mutagensis in E. coli. Conformational structuresand catalytic reaction kinetics of mutant enzymes were compared with wild type $\gamma$-GCS afterpurification. The S495F mutant enzyme (serine at 495 residue was substituted with phenylalanine),which had no catalytic activity for $\gamma$-glutamylcysteine synthesis, rarely folded even in neutral pH.However, the mutant A494V (alanine of 494 residue was replaced by valnine) which showed 50 %increase of activity, had a high folding structure. The folding structure of A494V also more stable athigh temperature and extreme pH compared to wild type and S495F. Reaction kinetics of wild typeand A494V were also investigated, Km value of A494V was smaller than that of wild type, while itshowed a little difference at Vmax values. This result evolved that alanine at 494 may be involved inbinding site of substrate rather than catalytic site. In addition, change of catalytic activity by onepoint mutation was highly correlated with the folding structure of enzyme.

• BMB Reports
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• 제41권2호
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• pp.126-131
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• 2008

The folding of aptamer immobilized on an Au electrode was successfully detected using label-free electrochemical methods. A thrombin binding DNA aptamer was used as a model system in the presence of various monovalent cations. Impedance spectra showed that the extent to which monovalent cations assist in folding of aptamer is ordered as $K^+$ > $NH_4^+$ > $Na^+$ > $Cs^+$. Our XPS analysis also showed that $K^+$ and $NH_4^+$ caused a conformational change of the aptamer in which it forms a stable complex with these monovalent ions. Impedance results for the interaction between aptamer and thrombin indicated that thrombin interacts more with folded aptamer than with unfolded aptamer. The EQCM technique provided a quantitative analysis of these results. In particular, the present impedance results showed that thrombin participates a folding of aptamer to some extent, and XPS analysis confirmed that thrombin stabilizes and induces the folding of aptamer.