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. Here we investigate the redox-coupled folding dynamics of equine heart cytochrome c (cyt-c) induced by external electron injection by using time-resolved X-ray solution scattering. A systematic kinetic analysis unveils a kinetic model for its folding with a stretched exponential behavior during the transition toward the folded state. With the aid of the ensemble optimization method combined with molecular dynamics simulations, we found that during the folding the heterogeneously populated ensemble of the unfolded state is converted to a narrowly populated ensemble of folded conformations. These observations obtained from the kinetic and the structural analyses of X-ray scattering data reveal that the folding dynamics of cyt-c accompanies many parallel pathways associated with the heterogeneously populated ensemble of unfolded conformations, resulting in the stretched exponential kinetics at room temperature. This finding provides direct evidence with a view to microscopic protein conformations that the cyt-c folding initiates from a highly heterogeneous unfolded state, passes through still diverse intermediate structures, and reaches structural homogeneity by arriving at the folded state.

Evidence for laser-induced homogeneous oriented ice nucleation revealed via pulsed x-ray diffraction.

The induction of homogeneous and oriented ice nucleation has to date not been achieved. Here, we report induced nucleation of ice from millimeter sized supercooled water drops illuminated by ns-optical laser pulses well below the ionization threshold making use of particular laser beam configurations and polarizations. Employing a 100 ps synchrotron x-ray pulse 100 ns after each laser pulse, an unambiguous correlation was observed between the directions and the symmetry of the laser fields and that of the H-bonding arrays of the induced ice crystals. Moreover, an analysis of the x-ray diffraction data indicates that, in the main, the induced nucleation of ice is homogeneous at temperatures well above the observed and predicted values for supercooled water.

Demonstration of a picosecond Bragg switch for hard X-rays in a synchrotron-based pump-probe experiment.

A benchmark experiment is reported that demonstrates the shortening of hard X-ray pulses in a synchrotron-based optical pump-X-ray probe measurement. The pulse-shortening device is a photoacoustic Bragg switch that reduces the temporal resolution of an incident X-ray pulse to approximately 7.5 ps. The Bragg switch is employed to monitor propagating sound waves in nanometer thin epitaxial films. From the experimental data, the pulse duration, diffraction efficiency and switching contrast of the device can be inferred. A detailed efficiency analysis shows that the switch can deliver up to 109 photons s-1 in high-repetition-rate synchrotron experiments.

Photochemical Mechanism of an Atypical Algal Phytochrome.

Phytochromes are bilin-containing photoreceptors that are typically sensitive to the red/far-red region of the visible spectrum. Recently, phytochromes from certain eukaryotic algae have become attractive targets for optogenetic applications because of their unique ability to respond to multiple wavelengths of light. Herein, a combination of time-resolved spectroscopy and structural approaches across picosecond to second timescales have been used to map photochemical mechanisms and structural changes in this atypical group of phytochromes. The photochemistry of an orange/far-red light-sensitive algal phytochrome from Dolihomastix tenuilepis has been investigated by using a combination of visible, IR and X-ray scattering probes. The entire photocycle, correlated with accompanying structural changes in the cofactor/protein, are reported. This study identifies a complex photocycle for this atypical phytochrome. It also highlights a need to combine outcomes from a range of biophysical approaches to unravel complex photochemical and macromolecular processes in multi-domain photoreceptor proteins that are the basis of biological light-mediated signalling.

Transient isomers in the photodissociation of bromoiodomethane.

The photochemistry of halomethanes is fascinating for the complex cascade reactions toward either the parent or newly synthesized molecules. Here, we address the structural rearrangement of photodissociated CH2IBr in methanol and cyclohexane, probed by time-resolved X-ray scattering in liquid solution. Upon selective laser cleavage of the C-I bond, we follow the reaction cascade of the two geminate geometrical isomers, CH2I-Br and CH2Br-I. Both meta-stable isomers decay on different time scales, mediated by solvent interaction, toward the original parent molecule. We observe the internal rearrangement of CH2Br-I to CH2I-Br in cyclohexane by extending the time window up to 3 µs. We track the photoproduct kinetics of CH2Br-I in methanol solution where only one isomer is observed. The effect of the polarity of solvent on the geminate recombination pathways is discussed.

Solvent-dependent structure of molecular iodine probed by picosecond X-ray solution scattering.

The effect of solute-solvent interaction on molecular structure and reaction dynamics has been a target of intense studies in solution-phase chemistry, but it is often challenging to characterize the subtle effect of solute-solvent interaction even for the simplest diatomic molecules. Since the I2 molecule has only one structural parameter and exhibits solvatochromism, it is a good model system for investigating the solvent dependence of the solute structure. By using X-rays as a probe, time-resolved X-ray liquidography (TRXL) can directly elucidate the structures of reacting molecules in solution and can thus determine the solvent-dependent structural change with atomic resolution. Here, by applying TRXL, we characterized the molecular structure of I2 in methanol and cyclohexane with sub-angstrom accuracy. Specifically, we found that the I-I bond length of I2 is longer in the polar solvent (methanol) by ∼0.2 Å than in nonpolar solvents (cyclohexane and CCl4). Density functional theory (DFT) using 22 explicit methanol molecules well reproduces the longer I-I bond of molecular iodine in methanol and reveals that the larger bond length originates from partial negative charge filled in an antibonding σ* orbital through solvent-to-solute charge transfer.

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. The TR-XAS transient spectra measured at the iodine L1 and L3 edges support the same conclusion. The present work demonstrates that the application of these two complementary time-resolved X-ray methods to the same system can provide a detailed understanding of the reaction mechanism.

Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography.

To understand how signaling proteins function, it is crucial to know the time-ordered sequence of events that lead to the signaling state. We recently developed on the BioCARS 14-IDB beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in protein crystals with near-atomic spatial resolution and 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein (PYP) following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore over 10 decades of time. The first of four major intermediates characterized in this study is highly contorted, with the pCA carbonyl rotated nearly 90° out of the plane of the phenolate. A hydrogen bond between the pCA carbonyl and the Cys69 backbone constrains the chromophore in this unusual twisted conformation. Density functional theory calculations confirm that this structure is chemically plausible and corresponds to a strained cis intermediate. This unique structure is short-lived (∼600 ps), has not been observed in prior cryocrystallography experiments, and is the progenitor of intermediates characterized in previous nanosecond time-resolved Laue crystallography studies. The structural transitions unveiled during the PYP photocycle include trans/cis isomerization, the breaking and making of hydrogen bonds, formation/relaxation of strain, and gated water penetration into the interior of the protein. This mechanistically detailed, near-atomic resolution description of the complete PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics.

Ultrafast switching of hard X-rays.

A new concept for shortening hard X-ray pulses emitted from a third-generation synchrotron source down to few picoseconds is presented. The device, called the PicoSwitch, exploits the dynamics of coherent acoustic phonons in a photo-excited thin film. A characterization of the structure demonstrates switching times of ≤ 5 ps and a peak reflectivity of ∼10(-3). The device is tested in a real synchrotron-based pump-probe experiment and reveals features of coherent phonon propagation in a second thin film sample, thus demonstrating the potential to significantly improve the temporal resolution at existing synchrotron facilities.

Structure and spin of the low- and high-spin states of Fe2+(phen)3 studied by x-ray scattering and emission spectroscopy.

The structure and spin of photoexcited Fe2+(phen)3 in water are examined by x-ray scattering and x-ray emission spectroscopy with 100 ps time resolution. Excitation of the low-spin (LS) ground state (GS) to the charge transfer state 1MLCT* leads to the formation of a high-spin (HS) state that returns to the GS in 725 ps. Density functional theory (DFT) predicts a Fe-N bond elongation in HS by 0.19 Å in agreement with the scattering data. The angle between the ligands increases by 5.4° in HS, which allows the solvent to get 0.33 Å closer to Fe in spite of the expansion of the molecule. The rise in solvent temperature from the return of photoproducts to the GS is dominated by the formation dynamics of HS, 1MLCT* → HS, which is followed by a smaller rise from the HS → GS transition. The latter agrees with the 0.61 eV energy gap E(HS)-E(LS) calculated by DFT. However, the temperature rise from the 1MLCT → HS transition is greater than expected, by a factor of 2.1, which is explained by the re-excitation of nascent HS* by the 1.2 ps pump pulse. This hypothesis is supported by optical spectroscopy measurements showing that the 1.2 ps long pump pulse activates the HS* → 5MLCT* channel, which is followed by the ultrafast return to HS* via intersystem crossing. Finally, the spins of the photoproducts are monitored by the Kß emission and the spectra confirm that the spins of LS and HS states are 0 and 2, respectively.

Filming the birth of molecules and accompanying solvent rearrangement.

Molecules are often born with high energy and large-amplitude vibrations. In solution, a newly formed molecule cools down by transferring energy to the surrounding solvent molecules. The progression of the molecular and solute-solvent cage structure during this fundamental process has been elusive, and spectroscopic data generally do not provide such structural information. Here, we use picosecond X-ray liquidography (solution scattering) to visualize time-dependent structural changes associated with the vibrational relaxation of I(2) molecules in two different solvents, CCl(4) and cyclohexane. The birth and vibrational relaxation of I(2) molecules and the associated rearrangement of solvent molecules are mapped out in the form of a temporally varying interatomic distance distribution. The I-I distance increases up to ~4 Å and returns to the equilibrium distance (2.67 Å) in the ground state, and the first solvation cage expands by ~1.5 Å along the I-I axis and then shrinks back accompanying the structural change of the I(2) molecule.

Introducing a standard method for experimental determination of the solvent response in laser pump, X-ray probe time-resolved wide-angle X-ray scattering experiments on systems in solution.

In time-resolved laser pump, X-ray probe wide-angle X-ray scattering experiments on systems in solution the structural response of the system is accompanied by a solvent response. The solvent response is caused by reorganization of the bulk solvent following the laser pump event, and in order to extract the structural information of the solute, the solvent response has to be treated. Methodologies capable of doing so include both theoretical modelling and experimental determination of the solvent response. In the work presented here, we have investigated how to obtain a reproducible solvent response-the solvent term-experimentally when applying laser pump, X-ray probe time-resolved wide-angle X-ray scattering. The solvent term describes difference scattering arising from the structural response of the solvent to changes in the hydrodynamic parameters: pressure, temperature and density. We present results based on NIR and dye mediated solvent heating, and demonstrate that the solvent response is independent of the heating method. The NIR heating is shown to be rendered unusable by higher order effects under certain experimental conditions, while the dye mediated solvent heating is demonstrated to exhibit first order behaviour with respect to the amount of energy deposited in the solution. We introduce a standardized method for recording solvent responses in laser pump, X-ray probe time-resolved X-ray wide-angle scattering experiments by using dye mediated solvent heating. Furthermore, we have generated a library of solvent terms, which can be used to describe the solvent term in any TRWAXS experiment, and made it available online.

Real-time tracking of CO migration and binding in the α and ß subunits of human hemoglobin via 150-ps time-resolved Laue crystallography.

We have developed the method of picosecond Laue crystallography and used this capability to probe ligand dynamics in tetrameric R-state hemoglobin (Hb). Time-resolved, 2 Å-resolution electron density maps of photolyzed HbCO reveal the time-dependent population of CO in the binding (A) and primary docking (B) sites of both α and ß subunits from 100 ps to 10 µs. The proximity of the B site in the ß subunit is about 0.25 Å closer to its A binding site, and its kBA rebinding rate (~300 µs-1) is six times faster, suggesting distal control of the rebinding dynamics. Geminate rebinding in the ß subunit exhibits both prompt and delayed geminate phases. We developed a microscopic model to quantitatively explain the observed kinetics, with three states for the α subunit and four states for the ß subunit. This model provides a consistent framework for interpreting rebinding kinetics reported in prior studies of both HbCO and HbO2.

Protein structural dynamics of photoactive yellow protein in solution revealed by pump-probe X-ray solution scattering.

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 µs 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 reconstructed 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.

Structural dynamics of 1,2-diiodoethane in cyclohexane probed by picosecond X-ray liquidography.

We investigate the structural dynamics of iodine elimination reaction of 1,2-diiodoethane (C(2)H(4)I(2)) in cyclohexane by applying time-resolved X-ray liquidography (TRXL). The TRXL technique combines structural sensitivity of X-ray diffraction and 100 ps time resolution of X-ray pulses from synchrotron and allows direct probing of transient structure of reacting molecules. From the analysis of time-dependent X-ray solution scattering patterns using global fitting based on DFT calculation and MD simulation, we elucidate the kinetics and structure of transient intermediates resulting from photodissociation of C(2)H(4)I(2). In particular, the effect of solvent on the reaction kinetics and pathways is examined by comparison with an earlier TRXL study on the same reaction in methanol. In cyclohexane, the C(2)H(4)I radical intermediate undergoes two branched reaction pathways, formation of C(2)H(4)I-I isomer and direct dissociation into C(2)H(4) and I, while only isomer formation occurs in methanol. Also, the C(2)H(4)I-I isomer has a shorter lifetime in cyclohexane by an order of magnitude than in methanol. The difference in the reaction dynamics in the two solvents is accounted for by the difference in solvent polarity. In addition, we determine that the C(2)H(4)I radical has a bridged structure, not a classical structure, in cyclohexane.

Modeling difference x-ray scattering observations from an integral membrane protein within a detergent micelle.

Time-resolved x-ray solution scattering (TR-XSS) is a sub-field of structural biology, which observes secondary structural changes in proteins as they evolve along their functional pathways. While the number of distinct conformational states and their rise and decay can be extracted directly from TR-XSS experimental data recorded from light-sensitive systems, structural modeling is more challenging. This step often builds from complementary structural information, including secondary structural changes extracted from crystallographic studies or molecular dynamics simulations. When working with integral membrane proteins, another challenge arises because x-ray scattering from the protein and the surrounding detergent micelle interfere and these effects should be considered during structural modeling. Here, we utilize molecular dynamics simulations to explicitly incorporate the x-ray scattering cross term between a membrane protein and its surrounding detergent micelle when modeling TR-XSS data from photoactivated samples of detergent solubilized bacteriorhodopsin. This analysis provides theoretical foundations in support of our earlier approach to structural modeling that did not explicitly incorporate this cross term and improves agreement between experimental data and theoretical predictions at lower x-ray scattering angles.

Deciphering Photochemical Reaction Pathways of Aqueous Tetrachloroauric Acid by X-ray Transient Absorption Spectroscopy.

Photolysis reaction pathways of [Au(III)Cl4]- in aqueous solution have been investigated by time-resolved X-ray absorption spectroscopy. Ultraviolet excitation directly breaks the Au-Cl bond in [Au(III)Cl4]- to form [Au(II)Cl3]- that becomes highly reactive within 79 ps. Disproportionation of [Au(II)Cl3]- generates [Au(I)Cl2]-, which is stable for ≤10 µs. In contrast, intense near-infrared lasers photolyze water to generate hydrated electrons, which then reduce [Au(III)Cl4]- to [Au(II)Cl3]- at 5 ns. Hydrated electrons further induce a chain reaction from [Au(II)Cl3]- to [Au(0)Cl]- by successively removing one Cl-. The zero-valency Au anions quickly polymerize and condense to form Au nanoparticles, which become the dominating product after 400 s. Our results reveal that the condensation of zero-valency Au starts with dimerization of gold clusters coordinated with chloride ions rather than direct condensation of pristine Au atoms.

Photoactivation of triosmium dodecacarbonyl at 400 nm probed with time-resolved X-ray liquidography.

The photoactivation mechanism of Os3(CO)12 at 400 nm is examined with time-resolved X-ray liquidography. The data reveal two pathways: the vibrational relaxation following an internal conversion to the electronic ground state and the ligand dissociation to form Os3(CO)11 with a ligand vacancy at the axial position.

Time-resolved WAXS reveals accelerated conformational changes in iodoretinal-substituted proteorhodopsin.

Time-resolved wide-angle x-ray scattering (TR-WAXS) is an emerging biophysical method which probes protein conformational changes with time. Here we present a comparative TR-WAXS study of native green-absorbing proteorhodopsin (pR) from SAR86 and a halogenated derivative for which the retinal chromophore has been replaced with 13-desmethyl-13-iodoretinal (13-I-pR). Transient absorption spectroscopy differences show that the 13-I-pR photocycle is both accelerated and displays more complex kinetics than native pR. TR-WAXS difference data also reveal that protein structural changes rise and decay an order-of-magnitude more rapidly for 13-I-pR than native pR. Despite these differences, the amplitude and nature of the observed helical motions are not significantly affected by the substitution of the retinal's C-20 methyl group with an iodine atom. Molecular dynamics simulations indicate that a significant increase in free energy is associated with the 13-cis conformation of 13-I-pR, consistent with our observation that the transient 13-I-pR conformational state is reached more rapidly. We conclude that although the conformational trajectory is accelerated, the major transient conformation of pR is unaffected by the substitution of an iodinated retinal chromophore.

Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering.

We demonstrate tracking of protein structural changes with time-resolved wide-angle X-ray scattering (TR-WAXS) with nanosecond time resolution. We investigated the tertiary and quaternary conformational changes of human hemoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. We also report data on optically induced tertiary relaxations of myoglobin and refolding of cytochrome c to illustrate the wide applicability of the technique. By providing insights into the structural dynamics of proteins functioning in their natural environment, TR-WAXS complements and extends results obtained with time-resolved optical spectroscopy and X-ray crystallography.