32 research outputs found
Cooperative protein structural dynamics of homodimeric hemoglobin linked to water cluster at subunit interface revealed by time-resolved X-ray solution scattering
Homodimeric hemoglobin (HbI) consisting of two subunits is a good model system for investigating the allosteric structural transition as it exhibits cooperativity in ligand binding. In this work, as an effort to extend our previous study on wild-type and F97Y mutant HbI, we investigate structural dynamics of a mutant HbI in solution to examine the role of well-organized interfacial water cluster, which has been known to mediate intersubunit communication in HbI. In the T72V mutant of HbI, the interfacial water cluster in the T state is perturbed due to the lack of Thr72, resulting in two less interfacial water molecules than in wild-type HbI. By performing picosecond time-resolved X-ray solution scattering experiment and kinetic analysis on the T72V mutant, we identify three structurally distinct intermediates (I1, I2, and I3) and show that the kinetics of the T72V mutant are well described by the same kinetic model used for wild-type and F97Y HbI, which involves biphasic kinetics, geminate recombination, and bimolecular CO recombination. The optimized kinetic model shows that the R-T transition and bimolecular CO recombination are faster in the T72V mutant than in the wild type. From structural analysis using species-associated difference scattering curves for the intermediates, we find that the T-like deoxy I3 intermediate in solution has a different structure from deoxy HbI in crystal. In addition, we extract detailed structural parameters of the intermediates such as E-F distance, intersubunit rotation angle, and heme-heme distance. By comparing the structures of protein intermediates in wild-type HbI and the T72V mutant, we reveal how the perturbation in the interfacial water cluster affects the kinetics and structures of reaction intermediates of HbI. © 2016 Author(s)1571sciescopu
Direct Observation of Cooperative Protein Structural Dynamics of Homodimeric Hemoglobin from 100 ps to 10 ms with Pump–Probe X-ray Solution Scattering
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 Scapharca inaequivalvis 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-1, I-2, and I-3) 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-1 intermediate is generated within 100 ps and transforms to the R-like I-2 intermediate with a time constant of 3.2 +/- 0.2 ns. Subsequently, the late, T-like I-3 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 mu s for the partially photolyzed form. For the mutant, the overall kinetics are accelerated, and the formation of the T-like I-3 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.1149sciescopu
SVD-aided pseudo principal-component analysis: A new method to speed up and improve determination of the optimum kinetic model from time-resolved data
Determination of the optimum kinetic model is an essential prerequisite for
characterizing dynamics and mechanism of a reaction. Here, we propose a simple
method, termed as singular value decomposition-aided pseudo principal-component
analysis (SAPPA), to facilitate determination of the optimum kinetic model from
time-resolved data by bypassing any need to examine candidate kinetic models. We
demonstrate the wide applicability of SAPPA by examining three different sets of
experimental time-resolved data and show that SAPPA can efficiently determine the
optimum kinetic model. In addition, the results of SAPPA for both time-resolved
X-ray solution scattering (TRXSS) and transient absorption (TA) data of the same
protein reveal that global structural changes of protein, which is probed by TRXSS,
may occur more slowly than local structural changes around the chromophore,
which is probed by TA spectroscopy. VC 2017 Author(s). All article content, except
where otherwise noted, is licensed under a Creative Commons Attribution (CC BY)
license (http://creativecommons.org/licenses/by/4.0/).
[http://dx.doi.org/10.1063/1.4979854]1451sciescopu
Ultrafast X-ray crystallography and liquidography
Time-resolved X-ray diffraction provides direct information on three-dimensional structures of reacting molecules and thus can be used to elucidate structural dynamics of chemical and biological reactions. In this review, we discuss time-resolved X-ray diffraction on small molecules and proteins with particular emphasis on its application to crystalline (crystallography) and liquid-solution (liquidography) samples. Time-resolved X-ray diffraction has been used to study picosecond and slower dynamics at synchrotrons and can now access even femtosecond dynamics with the recent arrival of X-ray free-electron lasers.
(c) 2017 by Annual Reviews.All rights reserved2
Charge Transfer-Induced Torsional Dynamics in the Excited State of 2,6-Bis(diphenylamino)anthraquinone
Intramolecular charge transfer (ICT) in a multibranched push-pull chromophore is a key photophysical process which is attracting attention due to its relevance to the development of highly efficient organic light-emitting diodes, but the excited-state dynamics of multibranched push-pull chromophores is still unclear. Here, using femtosecond transient absorption spectroscopy and singular value decomposition analysis, we studied the excited state dynamics of 2,6-bis(diphenylamino)anthraquinone (DPA-AQ-DPA), which contains two diphenylamino (DPA) groups as electron-donors (D) and anthraquinone (AQ) as an electron-acceptor (A) and is a candidate for an efficient red TADF (thermally activated delayed fluorescence) emitter. The emission of DPA-AQ-DPA exhibits large Stokes shifts with increasing solvent polarity, indicating that the emission can be attributed to an ICT process. The charge separated (CS) state formed by ICT undergoes torsional dynamics, involving twisting between D and A, resulting in the formation of a twisted charge separated state (CStwisting). This twisting reaction between D and A is accelerated in high-polarity solvents compared with that in low-polarity solvents. Such faster CT-induced torsional dynamics in high-polarity solvents is explained in terms of the localization of ICT on one of two ICT branches, suggesting that DPA-AQ-DPA in localized CStwisting formed in high-polarity solvents has two different dihedral angles between a single A group and two D groups. On the other hand, with increasing solvent polarity, the CS and CStwisting states of DPA-AQ-DPA become stabilized, making their energy levels considerably lower than that of 3(π,π∗), consequently blocking the formation of the triplet excited state and TADF in a high-polarity solvent such as acetonitrile. By contrast, the energy levels of CS and CStwisting states in a low-polarity solvent, such as diethyl ether, are higher than that of 3(π,π∗), allowing for deactivation into 3DPA-AQ-DPA∗ through intersystem crossing. This result indicates that the energy levels of CS and CStwisting states can be adjusted by controlling aspects of the local environment, such as solvents, so that intersystem crossing can be either inhibited or promoted. In other words, the energy gap (ΔEST) between the lowest singlet and triplet excited states for DPA-AQ-DPA can be regulated by changing the solvent polarity. © 2017 American Chemical Society1111sciescopu
Direct Observation of Myoglobin Structural Dynamics from 100 picoseconds to 1 microsecond with Picosecond X-ray Solution Scattering
Here we report structural dynamics of equine myoglobin (Mb) in response to the CO photodissociation visualized by picosecond time-resolved X-ray solution scattering. The data clearly reveal new structural dynamics that occur in the timescale of similar to 360 picoseconds (ps) and similar to 9 nanoseconds (ns), which have not been clearly detected in previous studies.1128sciescopu
Conformational Substates of Myoglobin Intermediate Resolved by Picosecond X-ray Solution Scattering
Conformational substates of proteins are generally considered to play important roles in regulating protein functions, but an understanding of how they influence the structural dynamics and functions of the proteins has been elusive. Here, we investigate the structural dynamics of sperm whale myoglobin associated with the conformational substates using picosecond X-ray solution scattering. By applying kinetic analysis considering all of the plausible candidate models, we establish a kinetic model for the entire cycle of the protein transition in a wide time range from 100 ps to 10 ms. Four structurally distinct intermediates are formed during the cycle, and most importantly, the transition from the first intermediate to the second one (B -> C) occurs biphasically. We attribute the biphasic kinetics to the involvement of two conformational substates of the first intermediate, which are generated by the interplay between the distal histidine and the photodissociated CO.1115sciescopu
Charge Transfer-Induced Torsional Dynamics in the Excited State of 2,6-Bis(diphenylamino)anthraquinone
Intramolecular charge transfer (ICT) in a multibranched push–pull
chromophore is a key photophysical process which is attracting attention
due to its relevance to the development of highly efficient organic
light-emitting diodes, but the excited-state dynamics of multibranched
push–pull chromophores is still unclear. Here, using femtosecond
transient absorption spectroscopy and singular value decomposition
analysis, we studied the excited state dynamics of 2,6-bis(diphenylamino)anthraquinone
(DPA-AQ-DPA), which contains two diphenylamino (DPA) groups as electron-donors
(D) and anthraquinone (AQ) as an electron-acceptor (A) and is a candidate
for an efficient red TADF (thermally activated delayed fluorescence)
emitter. The emission of DPA-AQ-DPA exhibits large Stokes shifts with
increasing solvent polarity, indicating that the emission can be attributed
to an ICT process. The charge separated (CS) state formed by ICT undergoes
torsional dynamics, involving twisting between D and A, resulting
in the formation of a twisted charge separated state (CS<sub>twisting</sub>). This twisting reaction between D and A is accelerated in high-polarity
solvents compared with that in low-polarity solvents. Such faster
CT-induced torsional dynamics in high-polarity solvents is explained
in terms of the localization of ICT on one of two ICT branches,
suggesting that DPA-AQ-DPA in localized CS<sub>twisting</sub> formed
in high-polarity solvents has two different dihedral angles between
a single A group and two D groups. On the other hand, with increasing
solvent polarity, the CS and CS<sub>twisting</sub> states of DPA-AQ-DPA
become stabilized, making their energy levels considerably lower than
that of <sup>3</sup>(π,π*), consequently blocking the
formation of the triplet excited state and TADF in a high-polarity
solvent such as acetonitrile. By contrast, the energy levels of CS
and CS<sub>twisting</sub> states in a low-polarity solvent, such as
diethyl ether, are higher than that of <sup>3</sup>(π,π*),
allowing for deactivation into <sup>3</sup>DPA-AQ-DPA* through intersystem
crossing. This result indicates that the energy levels of CS and CS<sub>twisting</sub> states can be adjusted by controlling aspects of the
local environment, such as solvents, so that intersystem crossing
can be either inhibited or promoted. In other words, the energy gap
(Δ<i>E</i><sub>ST</sub>) between the lowest singlet
and triplet excited states for DPA-AQ-DPA can be regulated by changing
the solvent polarity
Conformational substates of myoglobin intermediate resolved by picosecond X-ray solution scattering
Conformational substates of proteins are generally considered to play important roles in regulating protein functions, but an understanding of how they influence the structural dynamics and functions of the proteins has been elusive. Here, we investigate the structural dynamics of sperm whale myoglobin associated with the conformational substates using picosecond X-ray solution scattering. By applying kinetic analysis considering all of the plausible candidate models, we establish a kinetic model for the entire cycle of the protein transition in a wide time range from 100 ps to 10 ms. Four structurally distinct intermediates are formed during the cycle, and most importantly, the transition from the first intermediate to the second one (B → C) occurs biphasically. We attribute the biphasic kinetics to the involvement of two conformational substates of the first intermediate, which are generated by the interplay between the distal histidine and the photodissociated CO. © 2014 American Chemical Society.114151sciescopu
Cooperative protein structural dynamics of homodimeric hemoglobin linked to water cluster at subunit interface revealed by time-resolved X-ray solution scattering
Homodimeric hemoglobin (HbI) consisting of two subunits is a good model system for investigating the allosteric structural transition as it exhibits cooperativity in ligand binding. In this work, as an effort to extend our previous study on wild-type and F97Y mutant HbI, we investigate structural dynamics of a mutant HbI in solution to examine the role of well-organized interfacial water cluster, which has been known to mediate intersubunit communication in HbI. In the T72V mutant of HbI, the interfacial water cluster in the T state is perturbed due to the lack of Thr72, resulting in two less interfacial water molecules than in wild-type HbI. By performing picosecond time-resolved X-ray solution scattering experiment and kinetic analysis on the T72V mutant, we identify three structurally distinct intermediates (I1, I2, and I3) and show that the kinetics of the T72V mutant are well described by the same kinetic model used for wild-type and F97Y HbI, which involves biphasic kinetics, geminate recombination, and bimolecular CO recombination. The optimized kinetic model shows that the R-T transition and bimolecular CO recombination are faster in the T72V mutant than in the wild type. From structural analysis using species-associated difference scattering curves for the intermediates, we find that the T-like deoxy I3 intermediate in solution has a different structure from deoxy HbI in crystal. In addition, we extract detailed structural parameters of the intermediates such as E-F distance, intersubunit rotation angle, and heme-heme distance. By comparing the structures of protein intermediates in wild-type HbI and the T72V mutant, we reveal how the perturbation in the interfacial water cluster affects the kinetics and structures of reaction intermediates of HbI