Search

Time-resolved small- and wide-angle X-ray scattering (SAXS/WAXS) studies of proteins in solution based on the pump-probe approach unveil structural information from intermediates over a broad range of length and time scales. In spite of the promise of this methodology, only a fraction of the wealth of information encoded in scattering data has been extracted in studies performed thus far. Here, we discuss the methodology, summarize results from recent time-resolved X-ray scattering studies, and examine the potential to extract additional information from these scattering curves.

The SARS‐CoV‐2 virus is highly contagious, as demonstrated by numerous well‐documented superspreading events. The infection commonly starts in the upper respiratory tract (URT) but can migrate to the lower respiratory tract (LRT) and other organs, often with severe consequences. Whereas LRT infection can lead to shedding of virus via breath and cough droplets, URT infection enables shedding via abundant speech droplets. Their viral load can be high in carriers with mild or no symptoms, an observation linked to the abundance of SARS‐CoV‐2‐susceptible cells in the oral cavity epithelium. Expelled droplets rapidly lose water through evaporation, with the smaller ones transforming into long‐lived aerosol. Although the largest speech droplets can carry more virions, they are few in number, fall to the ground rapidly and therefore play a relatively minor role in transmission. Of more concern is small speech aerosol, which can descend deep into the LRT and cause severe disease. However, since their total volume is small, the amount of virus they carry is low. Nevertheless, in closed environments with inadequate ventilation, they can accumulate, which elevates the risk of direct LRT infection. Of most concern is the large fraction of speech aerosol that is intermediate‐sized because it remains suspended in air for minutes and can be transported over considerable distances by convective air currents. The abundance of this speech‐generated aerosol, combined with its high viral load in pre‐ and asymptomatic individuals, strongly implicates airborne transmission of SARS‐CoV‐2 through speech as the primary contributor to its rapid spread.

Oral Fluid Droplets Generated during Speech In this experiment, investigators used a laser in a darkened box to visualize droplets that were generated during speech. The droplets decreased in numbe...

Correlated motions of proteins are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved X-ray methods hold promise for addressing this challenge but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature-jumps (T-jumps), through thermal excitation of the solvent, have been utilized to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform T-jump small- and wide-angle X-ray scattering (SAXS/WAXS) measurements on a dynamic enzyme, cyclophilin A (CypA), demonstrating that these experiments are able to capture functional intramolecular protein dynamics on the microsecond timescale. We show that CypA displays rich dynamics following a T-jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes. Two relaxation processes are resolved, a fast process is related to surface loop motions and slower process is related to motions in the core of the protein that are critical for catalytic turnover., Graphical Abstract

During a pandemic, identifying modes of transmission is paramount to devise effective and practical mitigation strategies. Mohamed Abbas and Didier Pittet1Abbas M Pittet D Surfing the COVID-19 scientific wave.Lancet Infect Dis. 2020; (published online June 30.)https://doi.org/10.1016/S1473-3099(20)30558-2Summary Full Text Full Text PDF Scopus (0) Google Scholar challenge the conclusions of our reports that normal speaking might be an important mode of transmission for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), involving small particles that remain airborne for minutes.2Anfinrud P Stadnytskyi V Bax CE Bax A Visualizing speech-generated oral fluid droplets with laser light scattering.N Engl J Med. 2020; 382: 2061-2063Crossref PubMed Scopus (50) Google Scholar, 3Stadnytskyi V Bax CE Bax A Anfinrud P The airborne lifetime of small speech droplets and their potential importance in SARS-CoV-2 transmission.Proc Natl Acad Sci USA. 2020; 117: 11875-11877Crossref PubMed Scopus (41) Google Scholar Whereas the opening remarks in Abbas and Pittet's correspondence are irrelevant to our work, we eagerly welcome an intellectual debate on the scientific merits of our research. In their correspondence, they claim that our “findings have no immediate implications”.1Abbas M Pittet D Surfing the COVID-19 scientific wave.Lancet Infect Dis. 2020; (published online June 30.)https://doi.org/10.1016/S1473-3099(20)30558-2Summary Full Text Full Text PDF Scopus (0) Google Scholar Nothing could be further from the truth. While we refer readers to the appendix (pp 1–5) for a detailed response to all issues raised, we here address two of Abbas and Pittet's more pertinent concerns.

This review focuses on how short X-ray pulses from synchrotrons and XFELs can be used to track light-induced structural changes in molecular complexes and proteins via the pump–probe method. The upgrade of the European Synchrotron Radiation Facility to a diffraction-limited storage ring, based on the seven-bend achromat lattice, and how it might boost future pump–probe experiments are described. We discuss some of the first X-ray experiments to achieve 100 ps time resolution, including the dissociation and in-cage recombination of diatomic molecules, as probed by wide-angle X-ray scattering, and the 3D filming of ligand transport in myoglobin, as probed by Laue diffraction. Finally, the use of femtosecond XFEL pulses to investigate primary chemical reactions, bond breakage and bond formation, isomerisation and electron transfer are discussed.

Speech droplets generated by asymptomatic carriers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are increasingly considered to be a likely mode of disease transmission. Highly sensitive laser light scattering observations have revealed that loud speech can emit thousands of oral fluid droplets per second. In a closed, stagnant air environment, they disappear from the window of view with time constants in the range of 8 to 14 min, which corresponds to droplet nuclei of ca. 4 μm diameter, or 12- to 21-μm droplets prior to dehydration. These observations confirm that there is a substantial probability that normal speaking causes airborne virus transmission in confined environments.

Speaking may be a primary mode of transmission of SARS-CoV-2. Considering that reports of asymptomatic transmission account for 50-80% of COVID-19 cases and that saliva has peak viral loads at time of patient presentation, droplet emission while speaking could be a significant factor driving transmission and warrants further study. We used a planar beam of laser light passing through a dust-free enclosure to detect saliva droplets emitted while speaking. We found that saying the words ‘Stay Healthy’ generates thousands of droplets that are otherwise invisible to the naked eye. A damp homemade cloth face mask dramatically reduced droplet excretion, with none of the spoken words causing a droplet rise above the background. Our preliminary findings have important implications for pandemic mitigation efforts.

Previous pressure-jump NMR experiments on a pressure-sensitized double mutant of ubiquitin showed evidence that its folding occurs via two parallel, comparably efficient pathways: A single barrier and a two-barrier pathway. An interrupted folding NMR experiment is introduced, where for a brief period the pressure is dropped to atmospheric conditions (1 bar), followed by a jump back to high pressure for signal detection. Conventional, forward sampling of the indirect dimension during the low-pressure period correlates the (15)N or (13)C′ chemical shifts of the unfolded protein at 1 bar to the (1)H frequencies of both the unfolded and folded proteins at high pressure. Remarkably, sampling the data of the same experiment in the reverse direction yields the frequencies of proteins present at the end of the low-pressure interval, which include unfolded, intermediate, and folded species. Although the folding intermediate (15)N shifts differ strongly from natively folded protein, its (13)C′ chemical shifts, which are more sensitive probes for secondary structure, closely match those of the folded protein and indicate that the folding intermediate must have a structure that is quite similar to the native state.

Pressure-jump hardware permits direct observation of protein NMR spectra during a cyclically repeated protein folding process. For a two-state folding protein, the change in resonance frequency will occur nearly instantaneously when the protein clears the transition state barrier, resulting in a mono-exponential change of the ensemble-averaged chemical shift. However, protein folding pathways can be more complex and contain meta-stable intermediates. With a pseudo-3D NMR experiment that utilizes stroboscopic observation, we measure the ensemble-averaged chemical shifts, including those of exchange-broadened intermediates, during the folding process. Such measurements for a pressure-sensitized mutant of ubiquitin show an on-pathway kinetic intermediate whose 15N chemical shifts differ most from the natively folded protein for strands β5, its preceding turn, and the two strands that pair with β5 in the native structure.

Brain tissue of Alzheimer’s disease patients invariably contains deposits of insoluble, fibrillar aggregates of peptide fragments of the amyloid precursor protein (APP), typically 40 or 42 residues in length and referred to as Aβ(40) and Aβ(42). However, it remains unclear whether these fibrils or oligomers constitute the toxic species. Depending on sample conditions, oligomers can form in a few seconds or less. These oligomers are invisible to solution NMR spectroscopy, but they can be rapidly (< 1 s) resolubilized and converted to their NMR-visible monomeric constituents by raising the hydrostatic pressure to a few kbar. Hence, utilizing pressure-jump NMR, the oligomeric state can be studied at residue-specific resolution by monitoring its signals in the monomeric state. Oligomeric states of Aβ(40) exhibit a high degree of order, reflected by slow longitudinal (15)N relaxation (T(1) >5 s) for residues 18–21 and 31–34, whereas the N-terminal 10 residues relax much faster (T(1) ≤1.5 s), indicative of extensive internal motions. Transverse relaxation rates rapidly increase to ca. 1000 s(−1) after the oligomerization is initiated.

It is well-known that tetrameric hemoglobin binds ligands cooperatively by undergoing a ligand-induced T → R quaternary structure transition, a structure-function relationship that has long served as a model system for understanding allostery in proteins. However, kinetic studies of the reverse, R → T quaternary structure transition following photolysis of carbonmonoxyhemoglobin (HbCO) reveal complex behavior that may be better explained by the presence of two different R quaternary structures coexisting in thermal equilibrium. Indeed, we report here time-resolved small- and wide-angle X-ray scattering (SAXS/WAXS) patterns of HbCO following a temperature jump that not only provide unambiguous evidence for more than one R state, but also unveil the time scale for interconversion between them. Since the time scale for the photolysis-induced R → T transition is likely different for different R-states, this structural heterogeneity must be accounted for to properly explain the kinetic heterogeneity observed in time-resolved spectroscopic studies following photolysis of HbCO.

Significance Development of specialized instrumentation enables rapid switching of the hydrostatic pressure inside an operating NMR spectrometer. This technology allows observation of protein signals during the repeated folding process. Applied to ubiquitin, a previously extensively studied model of protein folding, the methodology reveals an initially highly dynamic state that deviates relatively little from random coil behavior but also provides evidence for numerous repeatedly failed folding events, previously only observed in computer simulations. Above room temperature, direct NMR evidence shows a ∼50% fraction of proteins folding through an on-pathway kinetic intermediate, thereby revealing two equally efficient parallel folding pathways., In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. 15N and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15N relaxation data show evidence for rapid exchange, on a ∼100-μs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 μM, oligomeric off-pathway intermediates compete with folding of the native state.

A method is introduced that permits direct observation of the rates at which backbone amide hydrogens become protected from solvent exchange after rapidly dropping the hydrostatic pressure inside the NMR sample cell from denaturing (2.5 kbar) to native (1 bar) conditions. The method is demonstrated for a pressure-sensitized ubiquitin variant that contains two Val to Ala mutations. Increased protection against hydrogen exchange with solvent is monitored as a function of time during the folding process. Results for 53 backbone amides show narrow clustering with protection occurring with a time constant of ca 85 ms, but slower protection is observed around a reverse turn near the C-terminus of the protein. Remarkably, the native NMR spectrum returns with this slower time constant of ca 150 ms, indicating that the almost fully folded protein retains molten globule characteristics with severe NMR line broadening until the final hydrogen bonds are formed. Prior to crossing the transition state barrier, hydrogen exchange protection factors are close to unity, but with slightly elevated values in the β1-β2 hairpin, previously shown to be already lowly populated in the urea-denatured state.

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 A-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 β subunits from 100 ps to 10 μs. The proximity of the B site in the β subunit is about 0.25 A 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 β 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 β subunit. This model provides a consistent framework for interpreting rebinding kinetics reported in prior studies of both HbCO and HbO2.

The capacity to respond to environmental changes is crucial to an organism's survival. Halorhodospira halophila is a photosynthetic bacterium that swims away from blue light, presumably in an effort to evade photons energetic enough to be genetically harmful. The protein responsible for this response is believed to be photoactive yellow protein (PYP), whose chromophore photoisomerizes from trans to cis in the presence of blue light. We investigated the complete PYP photocycle by acquiring time-resolved small and wide-angle X-ray scattering patterns (SAXS/WAXS) over 10 decades of time spanning from 100 ps to 1 s. Using a sequential model, global analysis of the time-dependent scattering differences recovered four intermediates (pR0/pR1, pR2, pB0, pB1), the first three of which can be assigned to prior time-resolved crystal structures. The 1.8 ms pB0 to pB1 transition produces the PYP signaling state, whose radius of gyration (Rg = 16.6 A) is significantly larger than that for the ground state (Rg = 14.7 A) and is therefore inaccessible to time-resolved protein crystallography. The shape of the signaling state, reconstructed using GASBOR, is highly anisotropic and entails significant elongation of the long axis of the protein. This structural change is consistent with unfolding of the 25 residue N-terminal domain, which exposes the β-scaffold of this sensory protein to a potential binding partner. This mechanistically detailed description of the complete PYP photocycle, made possible by time-resolved crystal and solution studies, provides a framework for understanding signal transduction in proteins and for assessing and validating theoretical/computational approaches in protein biophysics.

BioCARS, a NIH-supported national user facility for macromolecular time-resolved X-ray crystallography at the Advanced Photon Source (APS), has recently completed commissioning of an upgraded undulator-based beamline optimized for single-shot laser-pump X-ray-probe measurements with time resolution as short as 100 ps. The source consists of two in-line undulators with periods of 23 and 27 mm that together provide high-flux pink-beam capability at 12 keV as well as first-harmonic coverage from 6.8 to 19 keV. A high-heat-load chopper reduces the average power load on downstream components, thereby preserving the surface figure of a Kirkpatrick–Baez mirror system capable of focusing the X-ray beam to a spot size of 90 µm horizontal by 20 µm vertical. A high-speed chopper isolates single X-ray pulses at 1 kHz in both hybrid and 24-bunch modes of the APS storage ring. In hybrid mode each isolated X-ray pulse delivers up to ∼4 × 1010photons to the sample, thereby achieving a time-averaged flux approaching that of fourth-generation X-FEL sources. A new high-power picosecond laser system delivers pulses tunable over the wavelength range 450–2000 nm. These pulses are synchronized to the storage-ring RF clock with long-term stability better than 10 ps RMS. Monochromatic experimental capability with Biosafety Level 3 certification has been retained.

We have developed a time-resolved x-ray scattering diffractometer capable of probing structural dynamics of proteins in solution with 100-ps time resolution. This diffractometer, developed on the ID14B BioCARS (Consortium for Advanced Radiation Sources) beamline at the Advanced Photon Source, records x-ray scattering snapshots over a broad range of q spanning 0.02–2.5 Å -1 , thereby providing simultaneous coverage of the small-angle x-ray scattering (SAXS) and wide-angle x-ray scattering (WAXS) regions. To demonstrate its capabilities, we have tracked structural changes in myoglobin as it undergoes a photolysis-induced transition from its carbon monoxy form (MbCO) to its deoxy form (Mb). Though the differences between the MbCO and Mb crystal structures are small (rmsd Å ), time-resolved x-ray scattering differences recorded over 8 decades of time from 100 ps to 10 ms are rich in structure, illustrating the sensitivity of this technique. A strong, negative-going feature in the SAXS region appears promptly and corresponds to a sudden > 22 Å 3 volume expansion of the protein. The ensuing conformational relaxation causes the protein to contract to a volume ∼2 Å 3 larger than MbCO within ∼10 ns. On the timescale for CO escape from the primary docking site, another change in the SAXS/WAXS fingerprint appears, demonstrating sensitivity to the location of the dissociated CO. Global analysis of the SAXS/WAXS patterns recovered time-independent scattering fingerprints for four intermediate states of Mb. These SAXS/WAXS fingerprints provide stringent constraints for putative models of conformational states and structural transitions between them.

A detailed mechanistic understanding of how a protein functions requires knowledge not only of its static structure, but also how its conformation evolves as it executes its function. The recent development of picosecond time-resolved X-ray crystallography has allowed us to visualize in real time and with atomic detail the conformational evolution of a protein. Here, we report the photolysis-induced structural evolution of wild-type and L29F myoglobin over times ranging from 100 ps to 3 μs. The sub-ns structural rearrangements that accompany ligand dissociation in wild-type and the mutant form differ dramatically, and lead to vastly different ligand migration dynamics. The correlated protein displacements provide a structural explanation for the kinetic differences. Our observation of functionally important protein motion on the sub-ns time scale was made possible by the 150-ps time resolution of the measurement, and demonstrates that picosecond dynamics are relevant to protein function. To visualize subtle structural changes without modeling, we developed a novel method for rendering time-resolved electron density that depicts motion as a color gradient across the atom or group of atoms that move. A sequence of these time-resolved images have been stitched together into a movie, which allows one to literally “watch” the protein as it executes its function.

We use time-resolved crystallography to observe the structural progression of a bacterial blue light photoreceptor throughout its photocycle. Data were collected from 10 ns to 100 ms after photoactivation of the E46Q mutant of photoactive yellow protein. Refinement of transient chromophore conformations shows that the spectroscopically distinct intermediates are formed via progressive disruption of the hydrogen bond network to the chromophore. Although structural change occurs within a few nanoseconds on and around the chromophore, it takes milliseconds for a distinct pattern of tertiary structural change to fully progress through the entire molecule, thus generating the putative signaling state. Remarkably, the coupling between the chromophore conformation and the tertiary structure of this small protein is not tight: there are leads and lags between changes in the conformation of the chromophore and the protein tertiary structure.

We present beamline ID09B that is designed for pump and probe experiments to 50 ps time-resolution. The beamline has been refurbished with a narrow-bandwidth undulator for Laue diffraction and diffraction from liquids. The new undulator has 235 poles, a 17 mm magnetic period and is operated at 6.5 mm gap. It produces a spectral flux of 2.0 × 108 photon/0.1% bw/pulse (10 mA) at the fundamental at 15.5 keV and an integral flux of 1.1 × 1010 photon pulse−1 in a 2.5% bandwidth. The optics has been renewed with a high-precision toroidal mirror and a cryogenic monochromator. The X-ray chopper used for single pulse selection is also described together with the femtosecond laser. Finally the diffraction from excited iodine molecules in CCl4 is investigated on the nanosecond time-scale. It turns out that the high-angle scattering is insensitive to the thermal chock from the laser; these oscillations are therefore readily used for structure determination. Conversely, the low-angle scattering probes the hydrodynamics of the liquid over longer length scales and the oscillations are believed to originate from thermal stress and expansion of the solvent.

Myoglobin has long served as a model system for understanding the relations between protein structure, dynamics, and function. Its ability to discriminate between toxic CO and vital O2, two small ligands that are almost equivalent in size and dipole moment, has attracted much attention. To understand discrimination and reversible ligand-binding in Mb, both the bound state and the "docked" state that leads to binding need to be studied. We have reported previously the nearly linear Fe–C–O geometry of bound CO and the nearly orthogonal geometry of docked CO [Lim et al. (1995), Science 269 : 962]. With the exception of X-ray structures, a preponderance of evidence points to a nearly linear Fe–C–O geometry and calls into question the proposal that the highly conserved distal histidine forces CO to bind in a nonoptimal geometry. The differences between the bound CO structures determined using IR and X-ray methods might arise from a water molecule hydrogen bonded to the distal histidine in some of the unit cells. Discrimination by Mb is manifested not only thermodynamically but also kinetically. Time-resolved CO rebinding studies that compare Mb with microperoxidase suggest that the heme pocket docking site in Mb exerts steric control of the ligand rebinding rate, slowing the rate of CO binding by a factor of more than 104.

The nature of ligand motion within proteins has been investigated by measuring femtosecond time-resolved infrared (IR) spectra of CO photodissociated from the haem of myoglobin. Upon dissociation, the CO rotates approximately 90 degrees and becomes trapped within a ligand docking site located near the binding site. Two trajectories, distinguished spectroscopically and kinetically with time constants of 0.20 +/- 0.05 ps and 0.52 +/- 0.10 ps, lead to CO located within the docking site with opposite orientations. The protein reorganizes about the "docked' CO with a time constant of 1.6 +/- 0.3 ps and quickly establishes an energetic barrier that inhibits the reverse rebinding process.

Time-resolved absorption spectra of Pr phytochrome were obtained using a regeneratively amplified femtosecond titanium : sapphire laser system. The early time transient absorption spectra are comprised of prompt Pr photobleaching, stimulated emission, and excited-state absorption features that decay with a 24 ps time constant that matches the ground state appearance time of the primary photoproduct. Based on the 5 ns radiative lifetime calculated from the absorption and spontaneous emission spectra and the fluorescence quantum yield of 5.5 (± 0.5) × 10−3, we calculate an excited-state lifetime of 28 ps that agrees well with the directly determined lifetime. The transient absorption spectra are consistent with a primary photochemical reaction quantum yield of 0.15, and the absorption spectrum of the primary photoproduct closely resembles that of the low-temperature trapped intermediate, lumi-R. We conclude that the primary photoisomerization, which is believed to be a Z,syn E,syn isomerization of the C15=C16 chromophore bond, occurs in 24 ps. © 1997 John Wiley & Sons, Inc. Biospectroscopy 3: 421–433, 1997

The intensity of single X-ray pulses from third generation synchrotrons like the ESRF, APS and Spring8 is now so high that it is feasible to conduct time resolved experiment with diffraction, scattering and spectroscopic techniques on samples ranging from small molecules to proteins, with 100 ps time resolution. This limit is dictated by the X-ray pulse length from a synchrotron. In a time-resolved X-ray experiment one first needs to initiate the process of interest in the sample and then open the X-ray shutter and record the signal as a function of time. In the experiments shown in this review, the signals are recorded on a large CCD detector in order to record the signal efficiently in space. Unfortunately, the time-resolution of current CCDs is still too slow, 100 μs at best, to be useful for monitoring molecular processes on molecular time scales. The only way to explore faster phenomena is to use the pump-probe method where the time resolution is obtained from varying the pump-probe delay. In the examples discussed here, the samples were excited by picosecond or nanosecond laser pulses and the excited samples were probed by 100 ps X-ray pulses from an undulator.

We have exploited the principle of photoselection and the method of time-resolved Small Angle X-ray Scattering (SAXS) to investigate protein size and shape changes following photoactivation of photoactive yellow protein (PYP) in solution with ~150 ps time resolution. This study partially overcomes the orientational average intrinsic to solution scattering methods, and provides structural information at a higher level of detail. Photoactivation of the p-coumaric acid (pCA) chromophore in PYP produces a highly-contorted, short-lived, red shifted intermediate (pR0), and triggers prompt, protein compaction of approximately 0.3% along the direction defined by the electronic transition dipole moment of the chromophore. Contraction along this dimension is accompanied by expansion along the orthogonal directions, with the net protein volume change being approximately -0.25%. More than half the strain arising from formation of pR0 is relieved by the pR0 to pR1 structure transition (1.8 ± 0.2 ns), with the persistent strain presumably contributing to the driving force needed to generate the spectroscopically blue-shifted pB signaling state. The results reported here are consistent with the near-atomic resolution structural dynamics reported in a recent time-resolved Laue crystallography study of PYP crystals, and suggest that the early-time structural dynamics in the crystalline state carry over to proteins in solution.

The dynamics of electronic and thermal relaxation of the heme in myoglobin (Mb) have been determined from subpicosecond time-resolved near-IR absorbance spectra of photoexcited Mb. A broad, short-lived absorbance feature near 12 000 cm-1 (833 nm) was observed and assigned to an excited electronic state of the heme. The photoexcited heme relaxes to its ground electronic state with a time constant of 3.4 ± 0.4 ps, leading to an absorbance spectrum that is indicative of a “hot” heme. The ensuing thermal relaxation was characterized by probing the spectral evolution of band III, a porphyrin−iron charge-transfer transition near 13 110 cm-1 (763 nm) that is sensitive to temperature. The temperature of the electronically relaxed heme cools exponentially with a time constant of 6.2 ± 0.5 ps. The implications of electronic and thermal relaxation to photolysis studies of liganded heme proteins are discussed.

The relative orientations of carbon monoxide (CO) bound to and photodissociated from myoglobin in solution have been determined with time-resolved infrared polarization spectroscopy. The bound CO is oriented < or = 7 degrees from the heme normal, corresponding to nearly linear FE-C-O. Upon dissociation from the Fe, CO becomes trapped in a docking site that orientationally constrains it to lie approximately in the plane of the heme. Because the bound and "docked" CO are oriented in nearly orthogonal directions CO binding from the docking site is suppressed. These solutions results help to establish how myoglobin discriminates against CO, a controversial issue dominated by the misconception that Fe-C-O is bent.

Time‐resolved mid‐ir absorption spectra of CO at 283 K have been measured 100 ps after photodissociation from human hemoglobin A, horse myoglobin, and sperm whale myoglobin. The spectra reveal two vibrational features that are narrower than any reported for CO in the condensed phase near room temperature, indicating that CO becomes localized in a rotationally constrained environment. The integrated absorbance under these narrow features is 0.53±0.05 times that found for sperm whale myoglobin in low temperature glasses. A model is developed that relates this reduction of integrated absorbance to molecular motion in a rotationally constrained environment. From this model, the barrier to CO rotation is found to be 1.5±0.25 kcal/mol. The two vibrational features are tentatively assigned to CO oriented oppositely in the same site within the heme pocket. Evidently, the residues circumscribing the heme pocket in hemoglobin and myoglobin fashion a cavity near the binding site that accommodates the dissociated CO and restricts its rotational motion. This ‘‘docking’’ site mediates ligand transport to and from the active binding site and may be important to the function of ligand‐binding heme proteins.

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.

Invertebrate hemoglobins, which range in size from dimers to assemblies of hundreds of subunits, offer excellent models for the investigation of allosteric protein function. A recurring theme among cooperative invertebrate hemoglobins is a subunit pairing involving the heme-embedding E and F helices, despite a lack of sequence conservation in these helices. This is quite different from the assembly of vertebrate hemoglobins and suggests that such a pairing is amenable for regulating oxygen delivery.We have carried out extensive time-resolved crystallographic analysis on the simplest of these hemoglobins, the cooperative homodimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis. Limited ligand-linked subunit rotation permits the full allosteric transition to be followed in crystals of HbI. These studies have been combined with mutant and functional studies along with conventional crystallographic analysis to provide a comprehensive understanding of the communication between subunits and the movement of ligands through the protein. Investigation of ligand migration through this protein suggests, surprisingly, that ligands primarily escape through a distal histidine gate, despite its involvement in the subunit interface. As a result, ligand escape may require transient subunit movement, which is inhibited in the protein crystal lattice. These findings emphasize the central role of dynamics in protein function.Our studies are also directed towards understanding cooperative protein function in larger invertebrate hemoglobin assemblies. Preliminary time-resolved crystallogaphic analysis on the tetrameric Scapharca HbII suggests similar structural transitions as HbI and offers the possibility of investigating how changes in one subunit directly impact a neighboring subunit in the heterodimeric halves of HbII. In addition, we will discuss new insights that may provide unifying themes for the basis of cooperativity among diverse invertebrate hemoglobins that form EF dimeric pairing.

The picosecond evolution of the tertiary conformation of myoglobin (Mb) after photodissociation of MbCO was investigated at room temperature by probing band III, a weak iron-porphyrin charge-transfer transition near 13,110 cm-1 (763 nm) that is sensitive to the out-of-plane displacement of the iron. Upon photolysis, the iron moves out of the plane of the porphyrin, causing a blue-shift of band III and a concomitant change in the protein conformation. The dynamics for this functionally important motion are highly nonexponential, in agreement with recent molecular dynamics simulations [Kuczera, K., Lambry, J.-C., Martin, J.-L. & Karplus, M. (1993) Proc. Natl. Acad. Sci. USA 90, 5805-5807]. The conformational change likely affects the height of the barrier to ligand rebinding and may explain nonexponential NO rebinding.

Contradictions in X-ray structures of intermediates in the photocycle of photoactive yellow protein

10 pages; International audience; A chopper system for time resolved pump-probe experiments with x-ray beams from a synchrotron is described. The system has three parts: a water-cooled heatload chopper, a high-speed chopper, and a millisecond shutter. The chopper system, which is installed in beamline ID09B at the European Synchrotron Radiation Facility, provides short x-ray pulses for pump-probe experiments with ultrafast lasers. The chopper system can produce x-ray pulses as short as 200 ns in a continuous beam and repeat at frequencies from 0 to 3 kHz. For bunch filling patterns of the synchrotron with pulse separations greater than 100 ns, the high-speed chopper can isolate single 100 ps x-ray pulses that are used for the highest time resolution. A new rotor in the high-speed chopper is presented with a single pulse (100 ps) and long pulse (10 μs) option. In white beam experiments, the heatload of the (noncooled) high-speed chopper is lowered by a heatload chopper, which absorbs 95% of the incoming power without affecting the pulses selected by the high speed chopper.

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.

To understand how proteins function, it is crucial to know the time-ordered sequence of structural changes that occur as they execute their designed function. We recently developed on the BioCARS beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in proteins with 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore [1], and geminate ligand-binding dynamics in hemoglobin [2]. Briefly, a picosecond laser pulse photoexcites a protein and triggers a structural change, which is probed with a suitably delayed picosecond X-ray pulse. This “pump-probe” approach recovers time-resolved diffraction “snapshots” whose corresponding electron density maps can be stitched together into a real-time movie of the structural changes that ensue. The mechanistically detailed, near-atomic resolution description of the PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics [3]. This research was supported in part by the Intramural Research Program of the NIH, NIDDK.References:[1] F. Schotte, H.S. Cho, V.R. Kaila, H. Kamikubo, N. Dashdorj, E.R. Henry, T. Graber, R. Henning, M. Wulff, G. Hummer, P.A. Anfinrud Proc. Natl. Acad. Sci. U.S.A. 109, 19256 (2012).[2] F. Schotte, H. S. Cho, J. Soman, M. Wulff, J. S. Olson and P. A. Anfinrud, Chemical Physics, 422, 98-106 (2013).[3] V.R.I. Kaila, F. Schotte, H.S. Cho, G. Hummer, and P.A. Anfinrud, Nature Chemistry, 6, 258 (2014).

Comparisons are given between the time evolution of vibrational and electronic spectra in condensed phases. The vibrational spectrum evolves on the time scale of vibrational dephasing which can be considerably longer than the available short laser pulses. Examples from the photodissociation of iron carbonyls are given.

The realization of solution phase pump‐probe diffraction experiments on beamline ID09B is described. The pink beam from a low‐K in‐vacuum undulator is used to study the structural dynamics of small molecules in solution to 100 picosecond time resolution and at atomic resolution. The X‐ray chopper and the associated timing modes of the synchrotron are described. The dissociation of molecular iodine in liquid CCl4 is studied by single pulse diffraction. The data probe not only the iodine structures but also the solvent structure as the latter is thermally excited by the flow of energy from recombining iodine atoms. The low‐q part of the diffraction spectra is a sensitive probe of the hydrodynamics of the solvent as a function of time.

Picosecond time-resolved crystallography was used to follow the dissociation of carbon monoxide from the heme pocket of a mutant sperm whale myoglobin and the resultant conformational changes. Electron-density maps have previously been created at various time points and used to describe amino-acid side-chain and carbon monoxide movements. In this work, difference refinement was employed to generate atomic coordinates at each time point in order to create a more explicit quantitative representation of the photo-dissociation process. After photolysis the carbon monoxide moves to a docking site, causing rearrangements in the heme-pocket residues, the coordinate changes of which can be plotted as a function of time. These include rotations of the heme-pocket phenylalanine concomitant with movement of the distal histidine toward the solvent, potentially allowing carbon monoxide movement in and out of the protein and proximal displacement of the heme iron. The degree of relaxation toward the intermediate and deoxy states was probed by analysis of the coordinate movements in the time-resolved models, revealing a non-linear progression toward the unbound state with coordinate movements that begin in the heme-pocket area and then propagate throughout the rest of the protein.

Work carried out over the last 30 years unveiled the role of structural dynamics in controlling protein function. Cavity networks modulate structural dynamics trajectories and are functionally relevant; in globins they have been assigned a role in ligand migration and docking. These findings raised renewed interest for time-resolved structural investigations of myoglobin (Mb), a simple heme protein displaying a photosensitive iron-ligand bond. Photodissociation of MbCO generates a nonequilibrium population of protein structures relaxing over a time range extending from picoseconds to milliseconds. This process triggers ligand migration to matrix cavities with clear-cut effects on the rate and yield of geminate rebinding. Here, we report subnanosecond time-resolved Laue diffraction data on the triple mutant YQR-Mb [Leu-29(B10)Tyr, His-64(E7)Gln, Thr-67(E10)Arg] that depict the sequence of structural events associated with heme and protein relaxation from 100 ps to 316 ns and above. The photodissociated ligand rapidly (29 )Phe or Leu( 29 )Trp suggests that the internal structure controls the rate and amplitude of the relaxation events. A correlation between structural dynamics as unveiled by Laue crystallography and functional properties of Mb is presented.

Time-resolved mid-IR spectroscopy combines molecular sensitivity with ultrafast capability to incisively probe protein-ligand interactions in model heme proteins. Highly conserved residues near the heme binding site fashion a ligand-docking site that mediates the transport of ligands to and from the binding site. We employ polarization anisotropy measurements to probe the orientation and orientational distribution of CO when bound to and docked near the active binding site, as well as the dynamics of ligand trapping in the primary docking site. In addition, we use more conventional transient absorption methods to probe the dynamics of ligand escape from this site, as well as the ultrafast dynamics of NO geminate recombination with the active binding site. The systems investigated include myoglobin, hemoglobin, and microperoxidase.

We have developed the method of picosecond time-resolved crystallography and used this technique to investigate structural dynamics in biological macromolecules at ambient temperature. Time-resolved snapshots of myoglobin following flash photolysis of the CO adduct were determined with 150 ps time resolution and < 2 A spatial resolution. The structures reveal numerous sites in which CO becomes transiently trapped, as well as correlated motion of the protein side chains. When a single point mutation was introduced in a position near the binding site (L29F), the departure of CO from the primary docking site was significantly accelerated. The dramatic differences in the correlated protein displacements provide a structural explanation for these kinetic differences.