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Dynamic Release of Bending Stress in Short dsDNA by Formation of a Kink and Forks
Kim, Cheolhee,Lee, O‐,chul,Kim, Jae‐,Yeol,Sung, Wokyung,Lee, Nam Ki WILEY‐VCH Verlag 2015 Angewandte Chemie Vol.54 No.31
<P><B>Abstract</B></P><P>Bending with high curvature is one of the major mechanical properties of double‐stranded DNA (dsDNA) that is essential for its biological functions. The emergence of a kink arising from local melting in the middle of dsDNA has been suggested as a mechanism of releasing the energy cost of bending. Herein, we report that strong bending induces two types of short dsDNA deformations, induced by two types of local melting, namely, a kink in the middle and forks at the ends, which we demonstrate using D‐shaped DNA nanostructures. The two types of deformed dsDNA structures dynamically interconvert on a millisecond timescale. The transition from a fork to a kink is dominated by entropic contribution (anti‐Arrhenius behavior), while the transition from a kink to a fork is dominated by enthalpic contributions. The presence of mismatches in dsDNA accelerates kink formation, and the transition from a kink to a fork is removed when the mismatch size is three base pairs.</P>
Regulation of Protein Structural Changes by Incorporation of a Small-Molecule Linker
Kim, Youngmin,Yang, Cheolhee,Kim, Tae Wu,Thamilselvan, Kamatchi,Kim, Yonggwan,Ihee, Hyotcherl MDPI 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.12
<P>Proteins have the potential to serve as nanomachines with well-controlled structural movements, and artificial control of their conformational changes is highly desirable for successful applications exploiting their dynamic structural characteristics. Here, we demonstrate an experimental approach for regulating the degree of conformational change in proteins by incorporating a small-molecule linker into a well-known photosensitive protein, photoactive yellow protein (PYP), which is sensitized by blue light and undergoes a photo-induced N-terminal protrusion coupled with chromophore-isomerization-triggered conformational changes. Specifically, we introduced thiol groups into specific sites of PYP through site-directed mutagenesis and then covalently conjugated a small-molecule linker into these sites, with the expectation that the linker is likely to constrain the structural changes associated with the attached positions. To investigate the structural dynamics of PYP incorporated with the small-molecule linker (SML-PYP), we employed the combination of small-angle X-ray scattering (SAXS), transient absorption (TA) spectroscopy and experiment-restrained rigid-body molecular dynamics (MD) simulation. Our results show that SML-PYP exhibits much reduced structural changes during photo-induced signaling as compared to wild-type PYP. This demonstrates that incorporating an external molecular linker can limit photo-induced structural dynamics of the protein and may be used as a strategy for fine control of protein structural dynamics in nanomachines.</P>
Direct characterization of protein oligomers and their quaternary structures by single-molecule FRET
Kim, Cheolhee,Kim, Jae Yeol,Kim, Seung Hyeon,Lee, Byung Il,Lee, Nam Ki The Royal Society of Chemistry 2012 Chemical communications Vol.48 No.8
<P>Using a single-molecule method, we directly distinguish among oligomers from monomers to tetramers and determine their quaternary structures. Using this method, we found that RecR forms a stable dimer and its oligomeric form is modulated by its own concentration and the interaction with RecO.</P> <P>Graphic Abstract</P><P>Protein oligomers from monomers to tetramers are directly distinguished by a single-molecule method. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c2cc16528g'> </P>
Kim, Tae Wu,Yang, Cheolhee,Kim, Youngmin,Kim, Jong Goo,Kim, Jeongho,Jung, Yang Ouk,Jun, Sunhong,Lee, Sang Jin,Park, Sungjun,Kosheleva, Irina,Henning, Robert,van Thor, Jasper J.,Ihee, Hyotcherl The Royal Society of Chemistry 2016 Physical chemistry chemical physics Vol.18 No.13
<P>Real-time probing of structural transitions of a photoactive protein is challenging owing to the lack of a universal time-resolved technique that can probe the changes in both global conformation and light-absorbing chromophores of the protein. In this work, we combine time-resolved X-ray solution scattering (TRXSS) and transient absorption (TA) spectroscopy to investigate how the global conformational changes involved in the photoinduced signal transduction of photoactive yellow protein (PYP) is temporally and spatially related to the local structural change around the light-absorbing chromophore. In particular, we examine the role of internal proton transfer in developing a signaling state of PYP by employing its E46Q mutant (E46Q-PYP), where the internal proton transfer is inhibited by the replacement of a proton donor. The comparison of TRXSS and TA spectroscopy data directly reveals that the global conformational change of the protein, which is probed by TRXSS, is temporally delayed by tens of microseconds from the local structural change of the chromophore, which is probed by TA spectroscopy. The molecular shape of the signaling state reconstructed from the TRXSS curves directly visualizes the three-dimensional conformations of protein intermediates and reveals that the smaller structural change in E46Q-PYP than in wild-type PYP suggested by previous studies is manifested in terms of much smaller protrusion, confirming that the signaling state of E46Q-PYP is only partially developed compared with that of wildtype PYP. This finding provides direct evidence of how the environmental change in the vicinity of the chromophore alters the conformational change of the entire protein matrix.</P>
High-deposition-rate position welding of Al 5083 alloy for spherical-type liquefied natural gas tank
Kim, Cheolhee,Ahn, Youngnam,Lee, Ki-Bae,Kim, Daesoon Professional Engineering Publishing Ltd. 2016 Proceedings of the Institution of Mechanical Engin Vol. No.
<P>High-deposition-rate welding is used in the fabrication of liquefied natural gas tanks from thick aluminium plates because the weld defect generally increases and the productivity decreases with increasing number of passes. In this study, high-deposition-rate gas metal arc welding was implemented in vertical-up and horizontal positions. High deposition was achieved in the vertical-up position by pulse welding using 2.4-mm single wire and 1.6-mm twin wires and in the horizontal position by electromagnetic lifting of the weld pool. The arc stabilities for the applied processes were examined and welding procedures for thick plates were developed. A total of 14 welding passes were required for the vertical-up welding of a 70-mm thick plate by using the conventional 1.6-mm single-wire process, and the fully penetrated welds could be achieved with 8 welding passes by using twin-wire welding. Also, 10 welding passes were required for the horizontal welding of a 35-mm-thick plate by the conventional approach, and 6-pass full-penetration welding was successfully implemented by applying an external electromagnetic field.</P>
Kim, Hyoun Sook,Im, Ha Na,An, Doo Ri,Yoon, Ji Young,Jang, Jun Young,Mobashery, Shahriar,Hesek, Dusan,Lee, Mijoon,Yoo, Jakyung,Cui, Minghua,Choi, Sun,Kim, Cheolhee,Lee, Nam Ki,Kim, Soon-Jong,Kim, Jin Y American Society for Biochemistry and Molecular Bi 2015 The Journal of biological chemistry Vol.290 No.41
<▼1><P><B>Background:</B> Csd6 is one of the cell shape-determining proteins in <I>H. pylori</I>.</P><P><B>Results:</B> The active site of Csd6 is tailored to function as an <SMALL>L</SMALL>,<SMALL>D</SMALL>-carboxypeptidase in the peptidoglycan-trimming process.</P><P><B>Conclusion:</B> Csd6 constitutes a new family of <SMALL>L</SMALL>,<SMALL>D</SMALL>-carboxypeptidase.</P><P><B>Significance:</B> The substrate limitation of Csd6 is a strategy that <I>H. pylori</I> uses to regulate its helical cell shape and motility.</P></▼1><▼2><P><I>Helicobacter pylori</I> causes gastrointestinal diseases, including gastric cancer. Its high motility in the viscous gastric mucosa facilitates colonization of the human stomach and depends on the helical cell shape and the flagella. In <I>H. pylori</I>, Csd6 is one of the cell shape-determining proteins that play key roles in alteration of cross-linking or by trimming of peptidoglycan muropeptides. Csd6 is also involved in deglycosylation of the flagellar protein FlaA. To better understand its function, biochemical, biophysical, and structural characterizations were carried out. We show that Csd6 has a three-domain architecture and exists as a dimer in solution. The N-terminal domain plays a key role in dimerization. The middle catalytic domain resembles those of <SMALL>L</SMALL>,<SMALL>D</SMALL>-transpeptidases, but its pocket-shaped active site is uniquely defined by the four loops I to IV, among which loops I and III show the most distinct variations from the known <SMALL>L</SMALL>,<SMALL>D</SMALL>-transpeptidases. Mass analyses confirm that Csd6 functions only as an <SMALL>L</SMALL>,<SMALL>D</SMALL>-carboxypeptidase and not as an <SMALL>L</SMALL>,<SMALL>D</SMALL>-transpeptidase. The <SMALL>D</SMALL>-Ala-complexed structure suggests possible binding modes of both the substrate and product to the catalytic domain. The C-terminal nuclear transport factor 2-like domain possesses a deep pocket for possible binding of pseudaminic acid, and <I>in silico</I> docking supports its role in deglycosylation of flagellin. On the basis of these findings, it is proposed that <I>H. pylori</I> Csd6 and its homologs constitute a new family of <SMALL>L</SMALL>,<SMALL>D</SMALL>-carboxypeptidase. This work provides insights into the function of Csd6 in regulating the helical cell shape and motility of <I>H. pylori</I>.</P></▼2>
Kim, Tae Wu,Lee, Jae Hyuk,Choi, Jungkweon,Kim, Kyung Hwan,van Wilderen, Luuk J.,Guerin, Laurent,Kim, Youngmin,Jung, Yang Ouk,Yang, Cheolhee,Kim, Jeongho,Wulff, Michael,van Thor, Jasper J.,Ihee, Hyotch American Chemical Society 2012 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY - Vol.134 No.6
<P>Photoreceptor proteins play crucial roles in receiving light stimuli that give rise to the responses required for biological function. However, structural characterization of conformational transition of the photoreceptors has been elusive in their native aqueous environment, even for a prototype photoreceptor, photoactive yellow protein (PYP). We employ pump probe X-ray solution scattering to probe the structural changes that occur during the photocycle of PYP in a wide time range from 3.16 mu s to 300 ms. By the analysis of both kinetics and structures of the intermediates, the structural progression of the protein in the solution phase is vividly visualized. We identify four structurally distinct intermediates and their associated five time constants and reconstructed the molecular shapes of the four intermediates from time-independent, species-associated difference scattering curves. The constructed structures of the intermediates show the large conformational changes such as the protrusion of N-terminus, which is restricted in the crystalline phase due to the crystal contact and thus could not be clearly observed by X-ray crystallography. The protrusion of the N-terminus and the protein volume gradually increase with the progress of the photocycle and becomes maximal in the final intermediate, which is proposed to be the signaling state. The data not only reveal that a common kinetic mechanism is applicable to both the crystalline and the solution phases, but also provide direct evidence for how the sample environment influences structural dynamics and the reaction rates of the PYP photocycle.</P>
Won, Cheolhee,Kim, Byung‐,Hak,Yi, Eun Hee,Choi, Kyung‐,Ju,Kim, Eun‐,Kyung,Jeong, Jong‐,Min,Lee, Jae‐,Ho,Jang, Ja‐,June,Yoon, Jung‐,Hwan,Jeong, Won‐,Il,P John Wiley and Sons Inc. 2015 Hepatology Vol.62 No.4
<P>Enhanced expression of the cancer stem cell (CSC) marker, CD133, is closely associated with a higher rate of tumor formation and poor prognosis in hepatocellular carcinoma (HCC) patients. Despite its clinical significance, the molecular mechanism underlying the deregulation of CD133 during tumor progression remains to be clarified. Here, we report on a novel mechanism by which interleukin‐6/signal transducer and activator of transcription 3 (IL‐6/STAT3) signaling up‐regulates expression of CD133 and promotes HCC progression. STAT3 activated by IL‐6 rapidly bound to CD133 promoter and increased protein levels of CD133 in HCC cells. Reversely, in hypoxic conditions, RNA interference silencing of STAT3 resulted in decrease of CD133 levels, even in the presence of IL‐6, with a concomitant decrease of hypoxia‐inducible factor 1 alpha (HIF‐1α) expression. Active STAT3 interacted with nuclear factor kappa B (NF‐κB) p65 subunit to positively regulate the transcription of HIF‐1α providing a mechanistic explanation on how those three oncogenes work together to increase the activity of CD133 in a hypoxic liver microenvironment. Activation of STAT3 and its consequent induction of HIF‐1α and CD133 expression were not observed in Toll‐like receptor 4/IL‐6 double‐knockout mice. Long‐term silencing of CD133 by a lentiviral‐based approach inhibited cancer cell‐cycle progression and suppressed <I>in vivo</I> tumorigenicity by down‐regulating expression of cytokinesis‐related genes, such as TACC1, ACF7, and CKAP5. We also found that sorafenib and STAT3 inhibitor nifuroxazide inhibit HCC xenograft formation by blocking activation of STAT3 and expression of CD133 and HIF‐1α proteins. <I>Conclusion</I>: IL‐6/STAT3 signaling induces expression of CD133 through functional cooperation with NF‐κB and HIF‐1α during liver carcinogenesis. Targeting STAT3‐mediated CD133 up‐regulation may represent a novel, effective treatment by eradicating the liver tumor microenvironment. (H<SMALL>EPATOLOGY</SMALL> 2015;62:1160‐1173)</P>