• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.86-86
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• 1997

Human annexin I is a member of annexin family of calcium dependent phospholipid binding proteins, which have been implicated in various physiological roles including phospholipase A$_2$ (PLA$_2$) inhibition, membrane fusion and calcium channel activity. In this work, the structure of N-terminally truncated human annexin I (Δ-annexin I) and its interactions with Ca$\^$2+/, ATP and cAMP were studied at atomic level by using $^1$H, $\^$15/N, $\^$l3/C NMR (nuclear magnetic resonance) spectroscopy. The effect of Ca$\^$2+/ binding on the structure of Δ-annexin I was investigated, and compared with that of Mg$\^$2+/ binding. The addition of Ca$\^$2+/ to Δ-annexin I caused some changes in the high field and low field regions of $^1$H NMR spectra. Whereas, upon addition of Mg$\^$2+/ to Δ-annexin I, almost no change could be observed. Also we found that the binding ratio of ATP to Δ-annexin I is 1. Because Δ-annexin I is a large protein with 35 kDa molecular weight, site-specific (carbonyl-$\^$l3/C, amide-$\^$15/N) labeling technique was used to determine the interaction sites of Δ-annexin I with Ca$\^$2+/ and ATP. Assignments of all the histidinyl carbonyl carbon resonances have been completed by using Δ-annexin I along with its specific 1,2-subdomain. The carbonyl carbon resonances originating from His52 and His246 of Δ-annexin I were significantly affected by Ca$\^$2+/ binding, and some Tyr and Phe resonances were also affected. The carbonyl carbon resonances originating from His52 is significantly affected by ATP binding, therefore His52 seems to be involved in the ATP binding site of Δ-annexin I.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.963-977
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• 2015

The well-characterized gram-positive bacterium Bacillus subtilis is an outstanding industrial candidate for protein expression owing to its single membrane and high capacity of secretion, simplifying the downstream processing of secretory proteins. During the last few years, there has been continuous progress in the illustration of secretion mechanisms and application of this robust host in various fields of life science, such as enzyme production, feed additives, and food and pharmaceutical industries. Here, we review the developments of Bacillus subtilis as a highly promising expression system illuminating strong chemical- and temperatureinducible and other types of promoters, strategies for ribosome-binding-site utilization, and the novel approach of signal peptide selection. Furthermore, we outline the main steps of the Sec pathway and the relevant elements as well as their interactions. In addition, we introduce the latest discoveries of Tat-related complex structures and functions and the countless applications of this full-folded protein secretion pathway. This review also lists some of the current understandings of ATP-binding cassette transporters. According to the extensive knowledge on the genetic modification strategies and molecular biology of Bacillus subtilis, we propose some suggestions and strategies for improving the yield of intended productions. We expect this to promote striking future developments in the optimization and application of this bacterium.

• Proceedings of the PSK Conference
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• 2001.10a
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• pp.299.2-300
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• 2001

• Journal of the Korean Magnetic Resonance Society
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• v.2 no.2
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• pp.141-151
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• 1998

Human annexin I is a member of annexin family of calcium dependent phospholipid binding proteins, which have been implicated in various physiological roles including phospholipase A2(PLA2) inhibition, membrane fusion and calcium channel activity. In this work, the structure of N-terminally truncated human annexin I ({{{{ DELTA }}-annexin I) and its interactions with Ca2+, ATP and cAMP were studied at atomic level by using nuclear magnetic resonance (NMR) spectroscopy. The effect of Ca2+ binding on the structure of {{{{ DELTA }}-annexin I was investigated. The addition of Ca2+ to {{{{ DELTA }}-annexin I caused some changes in 13C NMR spectra. Carbonyl carbon resonances of some histidines were significantly broadened by Ca2+ binding. However, in the case of methionine, phenylalanine, and tyrosin, small changes could be observed. We found that ATP and cAMP bind {{{{ DELTA }}-annexin I, and the binding ratio of ATP to {{{{ DELTA }}-annexin I is 1. These results are well consistent with the report that cAMP and ATP interact with annexin I, and affect the calcium channels formed by annexin I. Because {{{{ DELTA }}-annexin I is a large protein with 35 kDa molecular weight, site-specific (carbonyl-13C) labeling technique was used to study the interaction sites of {{{{ DELTA }}-annexin I with Ca2+. NMR study was focused on the carbonyl carbon resonances of tyrosine, phenylalanine, methionine and histidine residues of {{{{ DELTA }}-annexin I because the number of these amino acids is small in the amino acid sequence of {{{{ DELTA }}-annexin I.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.778-783
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• 2008

Anthrax is an infectious disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis. To identify the mitochondrial proteins that are expressed differently in murine macrophages infected with spores of B. anthracis Sterne, proteomic and MALDI-TOF/MS analyses of uninfected and infected macrophages were conducted. As a result, 13 mitochondrial proteins with different expression patterns were discovered in the infected murine macrophages, and some were identified as ATP5b, NIAP-5, ras-related GTP binding protein B isoform CRAa, along with several unnamed proteins. Among these proteins, ATP5b is related to energy production and cytoskeletal rearrangement, whereas NIAP-5 causes apoptosis of host cells due to binding with caspase-9. Therefore, this paper focused on ATP5b, which was found to be down regulated following infection. The downregulated ATP5b also reduced ATP production in the murine macrophages infected with B. anthracis spores. Consequently, this study represents the first mitochondrial proteome analysis of infected macrophages.

• Archives of Pharmacal Research
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• v.9 no.1
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• pp.29-38
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• 1986

The effects of ginseng saponins on the sarcolemmal $Na^{+}$, $K^{+}$-ATPase were compared to gypsophila saponin, sodium dodecylsulfate (SDS), and Triton X-100 to elucidate whether the effects are due to the membrane distruption, using a highly enriched preparation of cardiac sarcolemma prepared from dog ventricular myocardium. About 26% and 29% of vesicles in the preparation, enriched in ouabain-sensitive $Na^{+}$, $K^{+}$-ATP ase, $\beta$-adrenergic and muscarinic receptors are rightside-out and inside-out orientation, respectively. Ginseng saponins (triol>total> diol) inhibited $Na^{+}$, $K^{+}$-ATP ase activity, $Na^{+}$, $K^{+}$-ATPase activity and [$^{3}$H]ouabain binding of sarcolemmal vesicles. However, gypsophila saponin, SDS (0.4$\mu$g/$\mu$g protein) and Triton X-100 (0.6 $\mu$g/$\mu$g protein) caused about 1.35 and 1.40-fold increase in $Na^{+}$, $K^{+}$-ATPase activity and [$^{3}$H] oubain binding, respectively. Especially, the activating effect of gypsophila saponin on membrane Na+, K+ ATPase was detected at gypsophila saponin to sarcolemmal protein ratios as high as 100. Low dose of ginseng saponin (3$\mu$g/$\mu$g protein) decreased the phosphorylation sites and the concentration of ouabain binding sites (Bmax) without affecting the turnover number and affinity for ouabain binding, while gypsophila saponin, SDS(0.4 ug/ug protein), ahd Triton X-100 (0.6$\mu$g/$\mu$g protein) increased the Bmax. The results suggest that ginseng saponins cause a decrease in the number of active sites by interacting directly with $Na^{+}$, $K^{+}$-ATPase before disruption of membrane barriers of sarcolemmal vesicles.

• Proceedings of the PSK Conference
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• 2003.10b
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• pp.160.1-160.1
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• 2003

In order to confirm the biological function of ORF10 in cephabacin biosynthesis gene cluster of Lysobacter lactamgenus as an ATP-binding-cassette (ABC) transporter, the gene for ORF10 was amplified and subcloned into pET-28a(+) expression vector. After gene induction with 0.5 mM IPTG at 30~! and further cultivation at $30^~$ !. for 8 hr, a lot of the recombinant ORF10 protein was produced as soluble form in cytoplasmic fraction as well as a membrane protein in the membrane fraction as likely as other ABC transporters. (omitted)

• BMB Reports
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• v.38 no.3
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• pp.266-274
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• 2005

High temperature requirement A (HtrA) and its homologues constitute the HtrA familiy proteins, a group of heat shock-induced serine proteases. Bacterial HtrA proteins perform crucial functions with regard to protein quality control in the periplasmic space, functioning as both molecular chaperones and proteases. In contrast to other bacterial quality control proteins, including ClpXP, ClpAP, and HslUV, HtrA proteins contain no regulatory components or ATP binding domains. Thus, they are commonly referred to as ATP-independent chaperone proteases. Whereas the function of ATP-dependent chaperone-proteases is regulated by ATP hydrolysis, HtrA exhibits a PDZ domain and a temperature-dependent switch mechanism, which effects the change in its function from molecular chaperone to protease. This mechanism is also related to substrate recognition and the fine control of its function. Structural and biochemical analyses of the three HtrA proteins, DegP, DegQ, and DegS, have provided us with clues as to the functional regulation of HtrA proteins, as well as their roles in protein quality control at atomic scales. The objective of this brief review is to discuss some of the recent studies which have been conducted regarding the structure and function of these HtrA proteins, and to compare their roles in the context of protein quality control.

• Biomolecules & Therapeutics
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• v.9 no.2
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• pp.79-87
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• 2001

Heat shock proteins serve as chaperone by preventing the aggregation of denatured proteins and promote survival of pathogens in harsh environments. In this study, heat shock gene encoding a 84-kDa (p84) protein, which is one of the three major heat shock proteins in S. pneumoniae, was cloned and characterized. PCR with a forward primer derived from N-terminal amino acid sequence of the p84 and a reverse primer derived from the conserved second ATP-binding region of Clp family was used for amplification of the gene encoding the p84 and subsequently the PCR product was used for sequence determination. Sequence analysis of the p84 gene demonstrated that it is a member of ClpL. The deduced amino acid sequence of pneumococcal ClpL shows homology with other members of the Clp family, and particularly, even in variable leader region, with bovine Clp-like protein and L. lactis ClpL. S. pneumoniae clpL is the smallest clop member (701 amono acids) containing the two conserved ATP-binding regions, and hydrophilic N-terminal variable region of pneu-mococcal Clp ATPase is much shorter than any known Clp ATPases. Histidine tagged ClpL was overexpressed and purified from E. coli. Immunoblot analysis employing antisera raised against pneumococcus p84 demonstrated no cross-reactivity with Clp analog in Eschericha coli, Staphylococcus aureus and human HeLa cells. Preimmunization of mice with ClpL extended mice life partially but did not protect them from death.

• Proceedings of the Korean Biophysical Society Conference
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• 1996.07a
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• pp.24-24
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• 1996

SecA protein of E. coli is essential for the translocation of various precursor proteins across the plasma membrane. Along with it, SecA protein interacts with precursor proteins, SecY/E, SecB and is an ATPase which has multiple ATP binding sites. There is little known about the regulation mechanism of the protein translocation machinery. (omitted)