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Yang, Cheolhee,Choi, Minseo,Kim, Jong Goo,Kim, Hanui,Muniyappan, Srinivasan,Nozawa, Shunsuke,Adachi, Shin-ichi,Henning, Robert,Kosheleva, Irina,Ihee, Hyotcherl MDPI 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.11
<P>The quaternary transition between the relaxed (R) and tense (T) states of heme-binding proteins is a textbook example for the allosteric structural transition. Homodimeric hemoglobin (HbI) from <I>Scapharca inaequivalvis</I> is a useful model system for investigating the allosteric behavior because of the relatively simple quaternary structure. To understand the cooperative transition of HbI, wild-type and mutants of HbI have been studied by using time-resolved X-ray solution scattering (TRXSS), which is sensitive to the conformational changes. Herein, we review the structural dynamics of HbI investigated by TRXSS and compare the results of TRXSS with those of other techniques.</P>
Kim, Kyung Hwan,Muniyappan, Srinivasan,Oang, Key Young,Kim, Jong Goo,Nozawa, Shunsuke,Sato, Tokushi,Koshihara, Shin-ya,Henning, Robert,Kosheleva, Irina,Ki, Hosung,Kim, Youngmin,Kim, Tae Wu,Kim, Jeongh American Chemical Society 2012 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY - Vol.134 No.16
<P/><P>Proteins serve as molecular machines in performing their biological functions, but the detailed structural transitions are difficult to observe in their native aqueous environments in real time. For example, despite extensive studies, the solution-phase structures of the intermediates along the allosteric pathways for the transitions between the relaxed (R) and tense (T) forms have been elusive. In this work, we employed picosecond X-ray solution scattering and novel structural analysis to track the details of the structural dynamics of wild-type homodimeric hemoglobin (HbI) from the clam <I>Scapharca inaequivalvis</I> and its F97Y mutant over a wide time range from 100 ps to 56.2 ms. From kinetic analysis of the measured time-resolved X-ray solution scattering data, we identified three structurally distinct intermediates (I<SUB>1</SUB>, I<SUB>2</SUB>, and I<SUB>3</SUB>) and their kinetic pathways common for both the wild type and the mutant. The data revealed that the singly liganded and unliganded forms of each intermediate share the same structure, providing direct evidence that the ligand photolysis of only a single subunit induces the same structural change as the complete photolysis of both subunits does. In addition, by applying novel structural analysis to the scattering data, we elucidated the detailed structural changes in the protein, including changes in the heme–heme distance, the quaternary rotation angle of subunits, and interfacial water gain/loss. The earliest, R-like I<SUB>1</SUB> intermediate is generated within 100 ps and transforms to the R-like I<SUB>2</SUB> intermediate with a time constant of 3.2 ± 0.2 ns. Subsequently, the late, T-like I<SUB>3</SUB> intermediate is formed via subunit rotation, a decrease in the heme–heme distance, and substantial gain of interfacial water and exhibits ligation-dependent formation kinetics with time constants of 730 ± 120 ns for the fully photolyzed form and 5.6 ± 0.8 μs for the partially photolyzed form. For the mutant, the overall kinetics are accelerated, and the formation of the T-like I<SUB>3</SUB> intermediate involves interfacial water loss (instead of water entry) and lacks the contraction of the heme–heme distance, thus underscoring the dramatic effect of the F97Y mutation. The ability to keep track of the detailed movements of the protein in aqueous solution in real time provides new insights into the protein structural dynamics.</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>