• Bulletin of the Korean Chemical Society
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• 제30권2호
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• pp.289-290
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• 2009

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 1999년도 제39회 춘계학술발표대회
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• pp.48.1-48.1
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• 1999

• 한국자기공명학회논문지
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• 제18권2호
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• pp.58-62
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• 2014

Human transmembrane proteins (hTMPs) are closely related to transport, channel formation, signaling, cell to cell interaction, so they are the crucial target of modern medicinal drugs. In order to study the structure and function of these hTMPs, it is important to prepare reasonable amounts of proteins. However, their preparation is seriously difficult and time-consuming due to insufficient yields and low solubility of hTMPs. We tried to produce large amounts of Syndecan-4 transmembrane domain (Syd4-TM) that is related to the healing wounds and tumor for a long time. In this study, we performed the structure determination of Syd4-TM combining the Polarity Index at Slanted Angle (PISA) wheel pattern analysis based on $^{15}N-^1H$ 2D SAMPI-4 solid-state NMR of expressed Syd4-TM and Molecular Dynamics (MD) simulation using Discovery Studio 3.1.

• 한국자기공명학회논문지
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• 제14권2호
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• pp.88-104
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• 2010

Human melanocortin-4 receptor (hMC4R) has a critical role in part of energy homeostasis, and their heterozygous mutations related in genetic cause of severe human obesity. In order to study the structure and function of these membrane proteins, it is important to prepare the samples. However, the preparation of transmembrane peptide is seriously difficult and time-consuming. Overexpression and purification of membrane proteins was reported to be difficult due to their innate insoluble and toxic properties. Among the many difficulties, the most important is the difficulty in obtaining sufficient quantities of purified protein. Recently, we succeed to produce large amounts of the second transmembrane domain from the wild-type hMC4R (wt-TM2) and D90N mutant hMC4R (m-TM2) and proposed the structural difference of them in membrane-like environments. In this paper, we demonstrate the optimization procedures to express and purify wt-TM2 or m-TM2 peptides, and solution NMR studies in different detergents to get high-resolution spectra were also described.

• 한국자기공명학회논문지
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• 제21권3호
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• pp.90-95
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• 2017

The Z-DNA domain of human ADAR1 ($Z{\alpha}_{ADAR1}$) produces B-Z junction DNA through preferential binding to the CG-repeat segment and destabilizing the neighboring AT-rich region. However, this study could not answer the question of how many base-pairs in AT-rich region are destabilized by binding of $Z{\alpha}_{ADAR1}$. Thus, we have performed NMR experiments of $Z{\alpha}_{ADAR1}$ to the longer DNA duplex containing an 8-base-paired (8-bp) CG-repeat segment and a 12-bp AT-rich region. This study revealed that $Z{\alpha}_{ADAR1}$ preferentially binds to the CG-repeat segment rather than AT-rich region in a long DNA and then destabilizes at least 6 base-pairs in the neighboring AT-rich region for efficient B-Z transition of the CG-repeat segment.

• Bulletin of the Korean Chemical Society
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• 제34권12호
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• pp.3577-3585
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• 2013

Syndecan-4 (heparan sulfate proteoglycan), biologically important in cell-to-cell interactions and tumor suppression, was studied through mutation of the GXXXG motif of its transmembrane domain (Syd4-TM), a motif which governs dimerization. The expression and purification of the mutant (mSyd4-TM) were optimized here to assess the function of the GXXXG motif in the dimerization of Syd4-TM. mSyd4-TM was obtained in M9 minimal media and its oligomerization was identified by SDS PAGE, Circular Dichroism (CD) spectroscopy, mass spectrometry and NMR spectroscopy. The mutant, unlike Syd4-TM, did not form dimers and was observed as monomers. The GXXXG motif of Syd-4TM was shown to be an important structural determinant of its dimerization.

• Journal of Applied Biological Chemistry
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• 제50권3호
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• pp.109-114
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• 2007

Sar1p is a ras-related GTP-binding protein that functions in intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi complex. The effector domain of Ras family proteins is highly conserved and this domain is functionally interchangeable in plant, yeast and mammalian Sar1. Using a recombinant Brassica sar1 protein (Bsar1p) harboring point mutations in its effector domain, we here investigated the ability of Sar1p to bind and hydrolyze GTP and to interact with the two sar1-specific regulators, GTPase activating protein (GAP) and guanine exchange factor (GEF). The T51A and T55A mutations impaired Bsar1p intrinsic GTP-binding and GDP-dissociation activity. In contrast, mutations in the switch domain of Bsar1 did not affect its intrinsic GTPase activity. Moreover, the P50A, P54A, and S56A mutations affected the interaction between Bsar1p and GAP. P54A mutant protein did not interact with two regulating proteins, GEF and GAP, even though the mutation didn't affect the intrinsic GTP-binding, nucleotide exchange or GTPase activity of Bsar1p.

• 생명과학회지
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• 제16권6호
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• pp.1052-1059
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• 2006

다양한 종류의 박테리아에서부터 사람의 세포에 이르기까지 환경적인 스트레스나 병에 의한 스트레스 혹은 스트레스가 없는 상황에서도 열충격반응(heat shock response) 유도되어진다. 열충격반응에 노출된 세포에서는 모든 단백질의 발현이 정지되는 반면, 열충격단백질(heat shock proteins: HSPs)은 발현되어 스트레스로부터 세포를 보호한다. HSF1(heat shock factor 1)이라는 HSPs 유도단백질은 열충격반응시 단량체형태에서 삼중체의 형태로 구조변화를 일으켜 heat shock element(HSE)라고 불리우는 HSP gene의 발현 promoter에 특이적으로 결합하게 되어 HSPs를 발현시킨다. Human HSF1(hHSF1)은 다섯 개의 시스테인 잔기를 가지고 있는데 이 시스테인의 thiol(-SH)기는 강한 친전자성을 띔으로 급격히 산화되거나 질산화된다. 이러한 고찰은 시스테인 잔기가 산화 환원 의존적인 황산기/이황화결합 전환을 통해 구조적인 변화를 가져온다는 사실을 의미하고 있다. 따라서 본 연구에서는 여러 가지 산화환원제를 이용하여 HSF1에 존재하는 다섯 개의 시스테인 잔기의 역할과 삼량체 형성에 관여하는 잔기에 대하여 알아보고자 하였다. 또한 이황화결합을 통한 삼량체형성의 구조적변화의 관점에서 HSF1의 구조 변화와 DNA 결합력과의 상관관계에 관하여도 알아보고자 하였다. 본 연구결과로 HSF1의 DNA binding domain은 삼량체를 형성하는 구조적인 변화를 통해서 DNA에 대한 결합력이 증가되는 것을 알 수 있었는데 이것은 삼량체가 됨으로서 HSF1의 내부에 위치해 있던 DNA binding domain이 외부로 노출 되어져 DNA에 쉽게 결합할 수 있게 된다는 사실을 시사한다.

• 대한화학회지
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• 제67권5호
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• pp.348-352
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• 2023

p97, a universally conserved AAA+ ATPase, holds a central position in the ubiquitin-proteasome system, orchestrating myriad cellular activities with significant therapeutic implications. This protein primarily interacts with a diverse set of adaptor proteins through its N-terminal domain (NTD), which is structurally located at the periphery of the D1 hexamer ring. While there have been numerous structural elucidations of p97 complexed with adaptor proteins, the stoichiometry has remained elusive. In this work, we present the crystal structure of the p97-N/D1 hexamer bound to the FAF1-UBX domain at a resolution of 3.1 Å. Our findings reveal a 6:6 stoichiometry between the p97 hexamer and FAF1-UBX domain, deepening our understanding from preceding structural studies related to p97-NTD and UBX domain-containing proteins. These insights lay the groundwork for potential therapeutic interventions addressing cancer and neurodegenerative diseases.

• 한국자기공명학회논문지
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• 제20권2호
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• pp.56-60
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• 2016

Adenylate kinase (AK) enzyme which acts as the catalyst of reversible high energy phosphorylation reaction between ATP and AMP which associate with energetic metabolism and nucleic acid synthesis and signal transmission. This enzyme has three distinct domains: Core, AMP binding domain (AMPbd) and Lid domain (LID). The primary role of AMPbd and LID is associated with conformational changes due to flexibility of two domains. Three dimensional structure of human AK1 has not been confirmed and various mutation experiments have been done to determine the active sites. In this study, AK1R138A which is changed arginine[138] of LID domain with alanine[138] was made and conducted with NMR experiments, backbone dynamics analysis and mo-lecular docking dynamic simulation to find the cause of structural change and substrate binding site. Synthetic human muscle type adenylate kinase 1 (hAK1) and its mutant (AK1R138A) were re-combinded with E. coli and expressed in M9 cell. Expressed proteins were purified and finally gained at 0.520 mM hAK1 and 0.252 mM AK1R138A. Multinuclear multidimensional NMR experiments including HNCA, HN(CO)CA, were conducted for amino acid sequence analysis and signal assignments of $^1H-^{15}N$ HSQC spectrum. Our chemical shift perturbation data is shown LID domain residues and around alanine[138] and per-turbation value(0.22ppm) of valine[179] is consid-ered as inter-communication effect with LID domain and the structural change between hAK1 and AK1R138A.