Structural dynamics of protein-protein association involved in the light-induced transition of Avena sativa LOV2 protein.

The Light-oxygen-voltage-sensing domain (LOV) superfamily, found in enzymes and signal transduction proteins, plays a crucial role in converting light signals into structural signals, mediating various biological mechanisms. While time-resolved spectroscopic studies have revealed the dynamics of the LOV-domain chromophore's electronic structures, understanding the structural changes in the protein moiety, particularly regarding light-induced dimerization, remains challenging. Here, we utilize time-resolved X-ray liquidography to capture the light-induced dimerization of Avena sativa LOV2.

Light-induced protein structural dynamics in bacteriophytochrome revealed by time-resolved x-ray solution scattering.

Bacteriophytochromes (BphPs) are photoreceptors that regulate a wide range of biological mechanisms via red light-absorbing (Pr)-to-far-red light-absorbing (Pfr) reversible photoconversion. The structural dynamics underlying Pfr-to-Pr photoconversion in a liquid solution phase are not well understood. We used time-resolved x-ray solution scattering (TRXSS) to capture light-induced structural transitions in the bathy BphP photosensory module of .

Determining the charge distribution and the direction of bond cleavage with femtosecond anisotropic x-ray liquidography.

Energy, structure, and charge are fundamental quantities characterizing a molecule. Whereas the energy flow and structure change in chemical reactions are experimentally characterized, determining the atomic charges of a molecule in solution has been elusive, even for a triatomic molecule such as triiodide ion, I. Moreover, it remains to be answered how the charge distribution is coupled to the molecular geometry; which I-I bond, if two I-I bonds are unequal, dissociates depending on the electronic state.

Filming ultrafast roaming-mediated isomerization of bismuth triiodide in solution.

Roaming reaction, defined as a reaction yielding products via reorientational motion in the long-range region (3 - 8 Å) of the potential, is a relatively recently proposed reaction pathway and is now regarded as a universal mechanism that can explain the unimolecular dissociation and isomerization of various molecules. The structural movements of the partially dissociated fragments originating from the frustrated bond fission at the onset of roaming, however, have been explored mostly via theoretical simulations and rarely observed experimentally. Here, we report an investigation of the structural dynamics during a roaming-mediated isomerization reaction of bismuth triiodide (BiI) in acetonitrile solution using femtosecond time-resolved x-ray liquidography.

Effect of the abolition of intersubunit salt bridges on allosteric protein structural dynamics.

A salt bridge, one of the representative structural factors established by non-covalent interactions, plays a crucial role in stabilizing the structure and regulating the protein function, but its role in dynamic processes has been elusive. Here, to scrutinize the structural and functional roles of the salt bridge in the process of performing the protein function, we investigated the effects of salt bridges on the allosteric structural transition of homodimeric hemoglobin (HbI) by applying time-resolved X-ray solution scattering (TRXSS) to the K30D mutant, in which the interfacial salt bridges of the wild type (WT) are abolished. The TRXSS data of K30D are consistent with the kinetic model that requires one monomer intermediate in addition to three structurally distinct dimer intermediates (I, I, and I) observed in WT and other mutants.

Ultrafast coherent motion and helix rearrangement of homodimeric hemoglobin visualized with femtosecond X-ray solution scattering.

Ultrafast motion of molecules, particularly the coherent motion, has been intensively investigated as a key factor guiding the reaction pathways. Recently, X-ray free-electron lasers (XFELs) have been utilized to elucidate the ultrafast motion of molecules. However, the studies on proteins using XFELs have been typically limited to the crystalline phase, and proteins in solution have rarely been investigated.

Femtosecond X-ray Liquidography Visualizes Wavepacket Trajectories in Multidimensional Nuclear Coordinates for a Bimolecular Reaction.

ConspectusVibrational wavepacket motions on potential energy surfaces are one of the critical factors that determine the reaction dynamics of photoinduced reactions. The motions of vibrational wavepackets are often discussed in the interpretation of observables measured with various time-resolved vibrational or electronic spectroscopies but mostly in terms of the frequencies of wavepacket motions, which are approximated by normal modes, rather than the actual positions of the wavepacket. Although the time-dependent positions (that is, the trajectory) of wavepackets are hypothesized or drawn in imagined or calculated potential energy surfaces, it is not trivial to experimentally determine the trajectory of wavepackets, especially in multidimensional nuclear coordinates for a polyatomic molecule.

Protein folding from heterogeneous unfolded state revealed by time-resolved X-ray solution scattering.

One of the most challenging tasks in biological science is to understand how a protein folds. In theoretical studies, the hypothesis adopting a funnel-like free-energy landscape has been recognized as a prominent scheme for explaining protein folding in views of both internal energy and conformational heterogeneity of a protein. Despite numerous experimental efforts, however, comprehensively studying protein folding with respect to its global conformational changes in conjunction with the heterogeneity has been elusive.

Estimating signal and noise of time-resolved X-ray solution scattering data at synchrotrons and XFELs.

Elucidating the structural dynamics of small molecules and proteins in the liquid solution phase is essential to ensure a fundamental understanding of their reaction mechanisms. In this regard, time-resolved X-ray solution scattering (TRXSS), also known as time-resolved X-ray liquidography (TRXL), has been established as a powerful technique for obtaining the structural information of reaction intermediates and products in the liquid solution phase and is expected to be applied to a wider range of molecules in the future. A TRXL experiment is generally performed at the beamline of a synchrotron or an X-ray free-electron laser (XFEL) to provide intense and short X-ray pulses.

Effect of Occluded Ligand Migration on the Kinetics and Structural Dynamics of Homodimeric Hemoglobin.

Small molecules such as molecular oxygen, nitric oxide, and carbon monoxide play important roles in life, and many proteins require the transport of small molecules to and from the bulk solvent for their function. Ligand migration within a protein molecule is expected to be closely related to the overall structural changes of the protein, but the detailed and quantitative connection remains elusive. For example, despite numerous studies, how occluded ligand migration affects the kinetics and structural dynamics of the R-T transition remains unclear.

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 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.

Atomistic characterization of the active-site solvation dynamics of a model photocatalyst.

The interactions between the reactive excited state of molecular photocatalysts and surrounding solvent dictate reaction mechanisms and pathways, but are not readily accessible to conventional optical spectroscopic techniques. Here we report an investigation of the structural and solvation dynamics following excitation of a model photocatalytic molecular system [Ir(dimen)], where dimen is para-diisocyanomenthane. The time-dependent structural changes in this model photocatalyst, as well as the changes in the solvation shell structure, have been measured with ultrafast diffuse X-ray scattering and simulated with Born-Oppenheimer Molecular Dynamics.

Femtosecond X-ray solution scattering reveals that bond formation mechanism of a gold trimer complex is independent of excitation wavelength.

The [Au(CN)2 (-)]3 trimer in water experiences a strong van der Waals interaction between the d(10) gold atoms due to large relativistic effect and can serve as an excellent model system to study the bond formation process in real time. The trimer in the ground state (S0) exists as a bent structure without the covalent bond between the gold atoms, and upon the laser excitation, one electron in the antibonding orbital goes to the bonding orbital, thereby inducing the formation of a covalent bond between gold atoms. This process has been studied by various time-resolved techniques, and most of the interpretation on the structure and dynamics converge except that the structure of the first intermediate (S1) has been debated due to different interpretations between femtosecond optical spectroscopy and femtosecond X-ray solution scattering.

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.

Combined probes of X-ray scattering and optical spectroscopy reveal how global conformational change is temporally and spatially linked to local structural perturbation in photoactive yellow protein.

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.

Identifying the major intermediate species by combining time-resolved X-ray solution scattering and X-ray absorption spectroscopy.

Identifying the intermediate species along a reaction pathway is a first step towards a complete understanding of the reaction mechanism, but often this task is not trivial. There has been a strong on-going debate: which of the three intermediates, the CHI2 radical, the CHI2-I isomer, and the CHI2(+) ion, is the dominant intermediate species formed in the photolysis of iodoform (CHI3)? Herein, by combining time-resolved X-ray liquidography (TRXL) and time-resolved X-ray absorption spectroscopy (TR-XAS), we present strong evidence that the CHI2 radical is dominantly formed from the photolysis of CHI3 in methanol at 267 nm within the available time resolution of the techniques (∼20 ps for TRXL and ∼100 ps for TR-XAS). The TRXL measurement, conducted using the time-slicing scheme, detected no CHI2-I isomer within our signal-to-noise ratio, indicating that, if formed, the CHI2-I isomer must be a minor intermediate.

Protein structural dynamics revealed by time-resolved X-ray solution scattering.

One of the most important questions in biological science is how a protein functions. When a protein performs its function, it undergoes regulated structural transitions. In this regard, to better understand the underlying principle of a protein function, it is desirable to monitor the dynamic evolution of the protein structure in real time.

Direct observation of bond formation in solution with femtosecond X-ray scattering.

The making and breaking of atomic bonds are essential processes in chemical reactions. Although the ultrafast dynamics of bond breaking have been studied intensively using time-resolved techniques, it is very difficult to study the structural dynamics of bond making, mainly because of its bimolecular nature. It is especially difficult to initiate and follow diffusion-limited bond formation in solution with ultrahigh time resolution.

Sub-100-ps structural dynamics of horse heart myoglobin probed by time-resolved X-ray solution scattering.

Here we report sub-100-ps structural dynamics of horse heart myoglobin revealed by time-resolved X-ray solution scattering. By applying the time-slicing scheme to the measurement and subsequent deconvolution, we investigate the protein structural dynamics that occur faster than the X-ray temporal pulse width of synchrotrons (~100 ps). The singular value decomposition analysis of the experimental data suggests that two structurally distinguishable intermediates are formed within 100 ps.

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.

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.