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

Melting Domain Size and Recrystallization Dynamics of Ice Revealed by Time-Resolved X-ray Scattering

The phase transition between water and ice is ubiquitous and one of the most important phenomena in nature. Here, we performed time-resolved x-ray scattering experiments capturing the melting and recrystallization dynamics of ice. The ultrafast heating of ice I is induced by an IR laser pulse and probed with an intense x-ray pulse, which provided us with direct structural information on different length scales. From the wide-angle x-ray scattering (WAXS) patterns, the molten fraction, as well as the corresponding temperature at each delay, were determined. The small-angle x-ray scattering (SAXS) patterns, together with the information extracted from the WAXS analysis, provided the time-dependent change of the size and the number of the liquid domains. The results show partial melting (~13 %) and superheating of ice occurring at around 20 ns. After 100 ns, the average size of the liquid domains grows from about 2.5 nm to 4.5 nm by the coalescence of approximately six adjacent domains. Subsequently, we capture the recrystallization of the liquid domains, which occurs on microsecond timescales due to the cooling by heat dissipation and results to a decrease of the average liquid domain size

X-ray Free Electron Laser Studies of Electron and Phonon Dynamics of Graphene Adsorbed on Copper

We report optical pumping and X-ray absorption spectroscopy experiments at the PAL free electron laser that directly probe the electron dynamics of a graphene monolayer adsorbed on copper in the femtosecond regime. By analyzing the results with ab-initio theory we infer that the excitation of graphene is dominated by indirect excitation from hot electron-hole pairs created in the copper by the optical laser pulse. However, once the excitation is created in graphene, its decay follows a similar path as in many previous studies of graphene adsorbed on semiconductors, i e. rapid excitation of SCOPS (Strongly Coupled Optical Phonons) and eventual thermalization. It is likely that the lifetime of the hot electron-hole pairs in copper governs the lifetime of the electronic excitation of the graphene.Comment: 22 pages, 8 Figures including Supplementary Materia

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

The time scale of the quaternary structural changes in hemoglobin revealed using the transient grating technique

The quaternary structural transition between the R and T states of human hemoglobin was investigated using the transient grating technique. The results presented herein reveal that the quaternary structural change accompanied by the R-T transition occurs within a few microseconds. © the Owner Societies 20151331sciescopu

Proton Transfer of Guanine Radical Cations Studied by Time-Resolved Resonance Raman Spectroscopy Combined with Pulse Radiolysis

The oxidation of guanine (G) is studied by using transient absorption and time-resolved resonance Raman spectroscopies combined with pulse radiolysis. The transient absorption spectral change demonstrates that the neutral radical of G (G•(−H+)), generated by the deprotonation of G radical cation (G•+), is rapidly converted to other G radical species. The formation of this species shows the pH dependence, suggesting that it is the G radical cation (G•+)′ formed from the protonation at the N7 of G•(−H+). On one hand, most Raman bands of (G•+)′ are up-shifted relative to those of G, indicating the increase in the bonding order of pyrimidine (Pyr) and imidazole rings. The (G•+)′ exhibits the characteristic CO stretching mode at ∼1266 cm−1 corresponding to a C−O single bond, indicating that the unpaired electron in (G•+)′ is localized on the oxygen of the Pyr ring. © 2015 American Chemical Society6

Protein Structural Dynamics of Wild-Type and Mutant Homodimeric Hemoglobin Studied by Time-Resolved X-Ray Solution Scattering

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 Scapharca inaequivalvis 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. © 2018 by the authors

Regulation of Protein Structural Changes by Incorporation of a Small-Molecule Linker

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

Coherent oscillations in chlorosome elucidated by two-dimensional electronic spectroscopy

Chlorosomes are the most efficient photosynthetic light-harvesting complexes found in nature and consist of many bacteriochlorophyll (BChl) molecules self-assembled into supramolecular aggregates. Here we elucidate the presence and the origin of coherent oscillations in chlorosome at cryogenic temperature using 2D electronic spectroscopy. We observe coherent oscillations of multiple frequencies superimposed on the ultrafast amplitude decay of 2D spectra. Comparison of oscillatory features in the rephasing and nonrephasing 2D spectra suggests that an oscillation of 620 cm-1 frequency arises from electronic coherence. However, this coherent oscillation can be enhanced by vibronic coupling with intermolecular vibrations of BChl aggregate, and thus it might originate from vibronic coherence rather than pure electronic coherence. Although the 620 cm-1 oscillation dephases rapidly, the electronic (or vibronic) coherence may still take part in the initial step of energy transfer in chlorosome, which is comparably fast. © 2014 American Chemical Society.112131sciescopu