• BMB Reports
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• v.33 no.6
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• pp.531-536
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• 2000

Tropomyosin (TM) is an important actin binding protein involved in regulation of muscle contraction. Unacetylated striated tropomyosin failed to bind to actin whereas unacetylated smooth tropomyosin bound well to actin. It has been demonstrated that high actin affinity of unacetylated ${\alpha}-tropomyosin$ was ascribed to the carboxyl terminal amino acid residues. In order to define the role of the carboxyl terminal residues of tropomyosin molecule on actin binding, two mutant tropomyosins were constructed. TM11 is identical to the striated tropomyosin except that the carboxyl terminal last three amino acids was replaced with $^{282}NNM^{284}$ whereas in TM14 $^{276}HA^{277}$ was substituted with smooth specific $^{276}QT^{277}$. TM11 and TM14 were overproduced in Escherichia coli and analyzed for actin affinity. The apparent binding constants (Kapp) of unacetylated tropomyosins were $2.2{\times}10^6M^{-1}$ for sm9, $1.03{\times}10^6M^{-1}$ for TM14, $0.19{\times}10^6M^{-1}$ for TM11, $>0.1{\times}10^6M^{-1}$ for striated, respectively. This result indicated that higher actin affinity of the unacetylated smooth tropomyosin was primarily attributed to the presence of QT residues in the smooth sequence. In case of the Ala-Ser (AS) dipeptide extension of the amino terminus of tropomyosin, Kapp were $21.1{\times}10^6M^{-1}$ for AS-sm9, $8.0{\times}10^6M^{-1}$ for AS-11, $4.7{\times}10^6M^{-1}$ for AS-14, $3.8{\times}10^6M^{-1}$ for AS-striated. AS-TM11 showed considerably higher actin affinity than AS-TM14, implying that interaction of Ala-Ser of the amino terminus with the carboxyl terminal residues. Since Kapp of AS-TM11 was significantly lower than that of AS-sm9, the presence of QT might be required for restoration of high actin affinity of the smooth ${\alpha}-tropomyosin$. These results suggested that the carboxyl terminal amino acid residues Glutamine275-Threonine276 are important for actin affinity of the recombinant smooth ${\alpha}-tropomyosin$, particularly of unacetylated smooth ${\alpha}-tropomyosin$.

• Bulletin of the Korean Chemical Society
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• v.32 no.4
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• pp.1183-1186
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• 2011

The unique Br signature was utilized for C-terminal amino acid sequencing of model peptides. C-terminal carboxyl group was selectively derivatized in peptides, containing side chain carboxyl group, using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and Br was introduced using 4-bromophenylhydrazine hydrochloride (BPH) in a one pot reaction. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) tandem mass spectra were obtained carrying the Br signature in the y-series ions. The Br signature facilitated C-terminal sequencing and discrimination of C-terminal carboxyl groups in the free acid and amide forms.

• Journal of Life Science
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• v.6 no.3
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• pp.185-192
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• 1996

Bacteriophage T7 gene 2.5 protein, a single-stranded DNA binding protein, is required for T7 DNA replication, recombination, and repair. T7 gene 2.5 protein has two distinctive domains, DNA binding and C-terminal domain, directly involved in protein-protein interaction. Gene 2.5 protein participates in the DNA replication of Bacteriophage T7, which makes this protein essential for the T7 growth and DNA replication. What gene 2.5 protein makes important at T7 growth and DNA replication is its binding affinity to single-stranded DNA and the protein-protein important at T7 DNA replication proteins which are essential for the T7 DNA synthesis. We have constructed pGST2.5(WT) encoding the wild-type gene 2.5 protein and pGST2.5$\Delta $21C lacking C-terminal 21 amino acid residues. The purified GST-fusion proteins, GST2.5(WT) and GST2.5(WT)$\Delta$21C, were used for whether the carboxyl-terminal domain participates in the protein-protein interactions or not. GST2.5(WT) and GST2.5$\Delta$21C showed the difference in the protein-protein interaction. GST2.5(WT) interacted with T7 DNA polymerase and gene 4 protein, but GST2.5$\Delta$21C did not interact with either protein. Secondly, GST2.5(WT) interacts with gene 4 proteins (helicase/primase) but not GST2.5$\Delta$21C. these results proved the involvement of the carboxyl-terminal domain of gene 2.5 protein in the protein-protein interaction. We clearly conclude that carboxy-terminal domain of gene 2.5 protein is firmly involved in protein-protein interactions in T7 replication proteins.

• BMB Reports
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• v.30 no.6
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• pp.410-414
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• 1997

We purified a protein carboxyl O-methyltransferase (protein methylase II) from porcine spleen to homogeneity. The molecular weight of the porcine spleen protein methylase II (ps-PM II) was estimated to be 27,500 daltons on SDS-PAGE. Amino acid sequence of N-terminal 28 residues for ps-PM II was identified. Amino-terminal three amino acid residues of ps-PM II were deleted when compared to those of other protein carboxyl methytransferase. S-Adenosyl-L-homocysteine competitively inhibits ps-PM II with a K, value of $1.63{\times}10^{-7}M$. Myelin basic protein exhibited the highest methyl-accepting capacity among the proteins tested.

• Journal of Life Science
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• v.11 no.1
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• pp.1-6
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• 2001

In order to determine the role of the carboxyl terminal amino acid residues of unacetylated ${\alpha}$-tropomyosin in actin affinity two mutant tropomyosins were constructed by site-directed mutagenesis. TM16 was identical to the striated tropomyosin except that three amino acids in the carboxyl terminal end were altered to $^{282}TNM^{284}$ while in TM17 $^{282}TSI^{284}$ of the striated was replaced with$^{282}NSM^{284}$. TM16 and TM17 were overproduced in Escherichia coli and analyzed for actin affinity by comparing actin affinities of the striated and TM11 $^{282}NNM^{284}$). The apparent binding constants (Kapp) of unacetylated tropomyosins to actin were $5.1{\times}10^4M^{-1}$ for the striated, $1.1{\times}10^5M^{-1}$ for TM11, $1.09{\times}10^5M^{-1}$ for TM16, and $1.03{\times}10^5M^{-1}$ for TM17, respectively. Since the actin affinities of TM11, TM16, and TM17 were very similar, this result suggested that amino acid residues 282 and 283 were insignificant for acting affinity of unacetylated $\alpha$-tropomyosin. However, they all exhibited higher actin affinities than that of the striated, suggesting that Met residue at the carboxyl terminus of unacetylated smooth tropomyosin was rather important for actin affinity, presumably due to the nucleophilic nature of sulfur atom in Met residue.

• Proceedings of the Korean Biophysical Society Conference
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• 1996.07a
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• pp.11-11
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• 1996

The three-dimensional structure of the carboxyl-terminal domain of the E.coli RNA polymerase $\alpha$ subunit, which is regarded as the contact site for transcription activator proteins and the promoter UP element, was determined by NMR spectroscopy. Its compact structure of four helices and two long arms enclosing its hydrophobic core shows a folding topology distinct from those of other DNA-binding proteins. (omitted)

• Proceedings of the Korean Society of Applied Pharmacology
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• 1992.05a
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• pp.42-42
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• 1992

ras oncogene은 암조직이나 transformed human cell line에서 거의 공통적으로 발견되는 oncogene으로서 그 product인 p2l 단백질은 C-terminal 25개의 아미노산 외에는 거의 동일한 배열을 가지고 있는 매우 conservative한 단백질이며 C-terminal cysteine이 carboxy methylation되어 있고 또한 palmitic acid와 같은 long chain fatty acid도 결합되어 있다. 보고된 바에 의하면 p21 protein의 palmitation은 ras protein의 세포막에 대한 친화력을 유지시키며 이와 같은 친화력은 cell transforming activity의 기본요건으로 알려져 있다. 이와 같은 관점에서 볼때 p21 단백질의 C-terminal processing현상을 new drug target으로, 즉 p2l 단백질의 C-terminal processing을 억제하므로서 cell transforming activity를 저해 할 수 있을 것이므로 생체내에 존재하는 p21 단백질 C-terminal processing 억제제의 identification 및 purification은 항암제 연구와 밀접한 관계가 있다. 구체적으로 farnesyl-protein transferase inhibitor 혹은 carboxyl methyl inhibitor의 identification 및 purification은 이같은 목적을 달성 할 수 있는 가능성이 크다.

• Proceedings of the Korean Magnetic Resonance Society Conference
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• 2001.02a
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• pp.29-29
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• 2001

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.4
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• pp.2173-2179
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• 2013

Ubiquitin carboxyl-terminal hydrolase 37 (UCH37, also called UCHL5), a member of the deubiquitinating enzymes, can suppress protein degradation through disassembling polyubiquitin from the distal subunit of the chain. It has been proved that UCH37 can be activated by proteasome ubiqutin chain receptor Rpn13 and incorporation into the 19S complex. UCH37, which has been reported to assist in the mental development of mice, may play an important role in oncogenesis, tumor invasion and migration. Further studies will allow a better understanding of roles in cell physiology and pathology, embryonic development and tumor formation, hopefully providing support for the idea that UCH37 may constitute a new interesting target for the development of anticancer drugs.

• Journal of the Korean Magnetic Resonance Society
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• v.14 no.2
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• pp.105-116
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• 2010

SH3YL1, a novel protein containing one Src homology 3 domain at the carboxyl terminus was first detected in mouse anagen skin cDNA. This protein had a significant homology with YHRO 16c/Ysc 84, the yeast Src homology 3 domain-containing protein. The sequence identity was remarkable at the carboxyl and amino-terminal Src homology 3 domain, suggesting that the novel protein is a mouse homolog of the yeast protein and thus was termed as SH3YL1. SH3YL1 is composed of two domains, a DUF500 at N-termini and a SH3 domain at C-termini. In our study we cloned the SH3 domain in bacterial expression system in Escherichia coli using pET32a vector with TEV protease cleavage site and purified as a monomer using affinity chromatography. The N-terminal poly-Histidine tag was cleaved with TEV protease and target protein was used for backbone studies. Our study showed that SH3 domain primarily consists of $\beta$-sheet which is in consistence with previous result performed on the truncated SH3 domain of SH3YL1.