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RISS 인기검색어
- 검색키워드
- 저자 : van Thor, Jasper J. (검색결과 3 건)
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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>
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Ramachandran, Pradeep L.,Lovett, Janet E.,Carl, Patrick J.,Cammarata, Marco,Lee, Jae Hyuk,Jung, Yang Ouk,Ihee, Hyotcherl,Timmel, Christiane R.,van Thor, Jasper J. American Chemical Society 2011 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY - Vol.133 No.24
<P>The signaling state of the photoactive yellow protein (PYP) photoreceptor is transiently developed via isomerization of its blue-light-absorbing chromophore. The associated structural rearrangements have large amplitude but, due to its transient nature and chemical exchange reactions that complicate NMR detection, its accurate three-dimensional structure in solution has been elusive. Here we report on direct structural observation of the transient signaling state by combining double electron electron resonance spectroscopy (DEER), NMR, and time-resolved pump–probe X-ray solution scattering (TR-SAXS/WAXS). Measurement of distance distributions for doubly spin-labeled photoreceptor constructs using DEER spectroscopy suggests that the signaling state is well ordered and shows that interspin-label distances change reversibly up to 19 Å upon illumination. The SAXS/WAXS difference signal for the signaling state relative to the ground state indicates the transient formation of an ordered and rearranged conformation, which has an increased radius of gyration, an increased maximum dimension, and a reduced excluded volume. Dynamical annealing calculations using the DEER derived long-range distance restraints in combination with short-range distance information from <SUP>1</SUP>H–<SUP>15</SUP>N HSQC perturbation spectroscopy give strong indication for a rearrangement that places part of the N-terminal domain in contact with the exposed chromophore binding cleft while the terminal residues extend away from the core. Time-resolved global structural information from pump–probe TR-SAXS/WAXS data supports this conformation and allows subsequent structural refinement that includes the combined energy terms from DEER, NMR, and SAXS/WAXS together. The resulting ensemble simultaneously satisfies all restraints, and the inclusion of TR-SAXS/WAXS effectively reduces the uncertainty arising from the possible spin-label orientations. The observations are essentially compatible with reduced folding of the I<SUB>2</SUB>′ state (also referred to as the ‘pB’ state) that is widely reported, but indicates it to be relatively ordered and rearranged. Furthermore, there is direct evidence for the repositioning of the N-terminal region in the I<SUB>2</SUB>′ state, which is structurally modeled by dynamical annealing and refinement calculations.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/2011/jacsat.2011.133.issue-24/ja200617t/production/images/medium/ja-2011-00617t_0002.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ja200617t'>ACS Electronic Supporting Info</A></P>
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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>
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