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1.  Time-resolved structural studies at synchrotrons and X-ray free electron lasers: opportunities and challenges 
X-ray free electron lasers (XFELs) are potentially revolutionary X-ray sources because of their very short pulse duration, extreme peak brilliance and high spatial coherence, features that distinguish them from today’s synchrotron sources. We review recent time-resolved Laue diffraction and time-resolved wide angle X-ray scattering (WAXS) studies at synchrotron sources, and initial static studies at XFELs. XFELs have the potential to transform the field of time-resolved structural biology, yet many challenges arise in devising and adapting hardware, experimental design and data analysis strategies to exploit their unusual properties. Despite these challenges, we are confident that XFEL sources are poised to shed new light on ultrafast protein reaction dynamics.
doi:10.1016/j.sbi.2012.08.006
PMCID: PMC3520507  PMID: 23021004
2.  Opportunities and challenges for time-resolved studies of protein structural dynamics at X-ray free-electron lasers 
X-ray free-electron lasers (XFELs) are revolutionary X-ray sources. Their time structure, providing X-ray pulses of a few tens of femtoseconds in duration; and their extreme peak brilliance, delivering approximately 1012 X-ray photons per pulse and facilitating sub-micrometre focusing, distinguish XFEL sources from synchrotron radiation. In this opinion piece, I argue that these properties of XFEL radiation will facilitate new discoveries in life science. I reason that time-resolved serial femtosecond crystallography and time-resolved wide angle X-ray scattering are promising areas of scientific investigation that will be advanced by XFEL capabilities, allowing new scientific questions to be addressed that are not accessible using established methods at storage ring facilities. These questions include visualizing ultrafast protein structural dynamics on the femtosecond to picosecond time-scale, as well as time-resolved diffraction studies of non-cyclic reactions. I argue that these emerging opportunities will stimulate a renaissance of interest in time-resolved structural biochemistry.
doi:10.1098/rstb.2013.0318
PMCID: PMC4052859  PMID: 24914150
X-ray free-electron lasers; structural biology; serial femtosecond crystallography; time-resolved diffraction; time-resolved wide angle X-ray scattering
3.  Probing Anisotropic Structure Changes in Proteins with Picosecond Time-Resolved Small Angle X-ray Scattering 
The journal of physical chemistry. B  2013;117(49):15825-15832.
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.
doi:10.1021/jp407593j
PMCID: PMC4124878  PMID: 24125473
photoisomerization; signal transduction; time-resolved small-angle x-ray scattering; photoactive yellow protein
4.  Three-dimensional electron crystallography of protein microcrystals 
eLife  2013;2:e01345.
We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1–1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name ‘MicroED’, that may have wide applicability in structural biology.
DOI: http://dx.doi.org/10.7554/eLife.01345.001
eLife digest
X-ray crystallography has been used to work out the atomic structure of a large number of proteins. In a typical X-ray crystallography experiment, a beam of X-rays is directed at a protein crystal, which scatters some of the X-ray photons to produce a diffraction pattern. The crystal is then rotated through a small angle and another diffraction pattern is recorded. Finally, after this process has been repeated enough times, it is possible to work backwards from the diffraction patterns to figure out the structure of the protein.
The crystals used for X-ray crystallography must be large to withstand the damage caused by repeated exposure to the X-ray beam. However, some proteins do not form crystals at all, and others only form small crystals. It is possible to overcome this problem by using extremely short pulses of X-rays, but this requires a very large number of small crystals and ultrashort X-ray pulses are only available at a handful of research centers around the world. There is, therefore, a need for other approaches that can determine the structure of proteins that only form small crystals.
Electron crystallography is similar to X-ray crystallography in that a protein crystal scatters a beam to produce a diffraction pattern. However, the interactions between the electrons in the beam and the crystal are much stronger than those between the X-ray photons and the crystal. This means that meaningful amounts of data can be collected from much smaller crystals. However, it is normally only possible to collect one diffraction pattern from each crystal because of beam induced damage. Researchers have developed methods to merge the diffraction patterns produced by hundreds of small crystals, but to date these techniques have only worked with very thin two-dimensional crystals that contain only one layer of the protein of interest.
Now Shi et al. report a new approach to electron crystallography that works with very small three-dimensional crystals. Called MicroED, this technique involves placing the crystal in a transmission electron cryo-microscope, which is a fairly standard piece of equipment in many laboratories. The normal ‘low-dose’ electron beam in one of these microscopes would normally damage the crystal after a single diffraction pattern had been collected. However, Shi et al. realized that it was possible to obtain diffraction patterns without severely damaging the crystal if they dramatically reduced the normal low-dose electron beam. By reducing the electron dose by a factor of 200, it was possible to collect up to 90 diffraction patterns from the same, very small, three-dimensional crystal, and then—similar to what happens in X-ray crystallography—work backwards to figure out the structure of the protein. Shi et al. demonstrated the feasibility of the MicroED approach by using it to determine the structure of lysozyme, which is widely used as a test protein in crystallography, with a resolution of 2.9 Å. This proof-of principle study paves the way for crystallographers to study protein that cannot be studied with existing techniques.
DOI: http://dx.doi.org/10.7554/eLife.01345.002
doi:10.7554/eLife.01345
PMCID: PMC3831942  PMID: 24252878
electron crystallography; electron diffraction; electron cryomicroscopy (cryo-EM); microED; protein structure; microcrystals; None
5.  Infrared Study of Carbon Monoxide Migration among Internal Cavities of Myoglobin Mutant L29W 
Journal of Biological Physics  2002;28(2):163-172.
Myoglobin, a small globular heme protein that binds gaseous ligands such asO2, CO and NO reversibly at the heme iron, provides an excellent modelsystem for studying structural and dynamic aspects of protein reactions. Flashphotolysis experiments, performed over wide ranges in time and temperature, reveal a complex ligand binding reaction with multiple kinetic intermediates, resulting from protein relaxation and movements of the ligand within the protein. Our recent studies of carbonmonoxy-myoglobin (MbCO) mutant L29W, using time-resolved infrared spectroscopy in combination with x-ray crystallography, have correlated kinetic intermediates with photoproduct structures that are characterized by the CO residing in different internal protein cavities, so-called xenon holes. Here we have used Fourier transform infrared temperature derivative spectroscopy (FTIR-TDS) to further examine the role of internal cavities in the dynamics. Different cavities can be accessed by the CO ligands at different temperatures, and characteristic infrared absorption spectra have been obtained for the different locations of the CO ligand within the protein, enabling us to monitor ligand migration through the protein as well as conformational changes of the protein.
doi:10.1023/A:1019990522433
PMCID: PMC3456666  PMID: 23345766
FTIR spectroscopy; ligand binding; myoglobin; temperature derivative spectroscopy
6.  Exploring ribozyme conformational changes with X-ray crystallography 
Methods (San Diego, Calif.)  2009;49(2):87-100.
Relating three-dimensional fold to function is a central challenge in RNA structural biology. Toward this goal, X-ray crystallography has long been considered the “gold standard” for structure determinations at atomic resolution, although NMR spectroscopy has become a powerhouse in this arena as well. In the area of dynamics, NMR remains the dominant technique to probe the magnitude and timescales of molecular motion. Although the latter area remains largely unassailable by conventional crystallographic methods, inroads have been made on proteins using Laue radiation on timescales of ms to ns. Proposed ‘fourth generation’ radiation sources, such as free-electron X-ray lasers, promise ps- to fs-timescale resolution, and credible evidence is emerging that supports the feasibility of single molecule imaging. At present however, the preponderance of RNA structural information has been derived from timescale and motion insensitive crystallographic techniques. Importantly, developments in computing, automation and high-flux synchrotron sources have propelled the rapidity of ‘conventional’ RNA crystal structure determinations to timeframes of hours once a suitable set of phases is obtained. With a sufficient number of crystal structures, it is possible to create a structural ensemble that can provide insight into global and local molecular motion characteristics that are relevant to biological function. Here we describe techniques to explore conformational changes in the hairpin ribozyme, a representative non-protein-coding RNA catalyst. The approaches discussed include: (i) construct choice and design using prior knowledge to improve X-ray diffraction; (ii) recognition of long-range conformational changes; and (iii) use of single-base or single-atom changes to create ensembles. The methods are broadly applicable to other RNA systems.
doi:10.1016/j.ymeth.2009.06.003
PMCID: PMC2782588  PMID: 19559088
RNA crystallography; ribozyme; crystallization; RNA structure; crystallographic ensembles; alternate conformation; long-range motion; fold and function; difference Fourier; non-protein-coding RNA
7.  Future Directions of Structural Mass Spectrometry using Hydroxyl Radical Footprinting 
Journal of mass spectrometry : JMS  2010;45(12):1373-1382.
Hydroxyl radical protein footprinting coupled to mass spectrometry has been developed over the last decade and has matured to a powerful method for analyzing protein structure and dynamics. It has been successfully applied in the analysis of protein structure, protein folding, protein dynamics, and protein-protein and protein-DNA interactions. Using synchrotron radiolysis, exposures of proteins to a “white” x-ray beam for milliseconds provide sufficient oxidative modifications to surface amino acid side chains that can be easily detected and quantified by mass spectrometry. Thus, conformational changes in proteins or protein complexes can be examined using a time-resolved approach, which would be a valuable method for the study of macromolecular dynamics. In this review, we describe a new application of hydroxyl radical protein footprinting to probe the time evolution of the calcium-dependent conformational changes of gelsolin on the millisecond timescale. The data suggest a cooperative transition as multiple sites in different molecular sub-domains have similar rates of conformational change. These findings demonstrate that time-resolved protein footprinting is suitable for studies of protein dynamics that occur over periods ranging from milliseconds to seconds. In this review we also show how the structural resolution and sensitivity of the technology can be improved as well. The hydroxyl radical varies in its reactivity to different side chains by over two orders of magnitude, thus oxidation of amino acid side chains of lower reactivity are more rarely observed in such experiments. Here we demonstrate that selected reaction monitoring (SRM)-based method can be utilized for quantification of oxidized species, improving the signal to noise ratio. This expansion of the set of oxidized residues of lower reactivity will improve the overall structural resolution of the technique. This approach is also suggested as a basis for developing hypothesis driven structural mass spectrometry experiments.
doi:10.1002/jms.1808
PMCID: PMC3012749  PMID: 20812376
8.  MARTINI bead form factors for the analysis of time-resolved X-ray scattering of proteins 
Journal of Applied Crystallography  2014;47(Pt 4):1190-1198.
Form factors for X-ray scattering calculations from coarse-grained MARTINI protein models are derived. The reliability at different levels of coarse-graining is evaluated and weighed against the gain in computational speed of the coarser models.
Time-resolved small- and wide-angle X-ray scattering (SAXS and WAXS) methods probe the structural dynamics of proteins in solution. Although technologically advanced, these methods are in many cases limited by data interpretation. The calculation of X-ray scattering profiles is computationally demanding and poses a bottleneck for all SAXS/WAXS-assisted structural refinement and, in particular, for the analysis of time-resolved data. A way of speeding up these calculations is to represent biomolecules as collections of coarse-grained scatterers. Here, such coarse-graining schemes are presented and discussed and their accuracies examined. It is demonstrated that scattering factors coincident with the popular MARTINI coarse-graining scheme produce reliable difference scattering in the range 0 < q < 0.75 Å−1. The findings are promising for future attempts at X-ray scattering data analysis, and may help to bridge the gap between time-resolved experiments and their interpretation.
doi:10.1107/S1600576714009959
PMCID: PMC4119947  PMID: 25242909
X-ray solution scattering; proteins; coarse-graining; MARTINI; structural dynamics; small-angle X-ray scattering (SAXS); wide-angle X-ray scattering (WAXS); protein structure refinement
9.  The kinetic dose limit in room-temperature time-resolved macromolecular crystallography 
Journal of Synchrotron Radiation  2012;19(Pt 2):264-273.
The effect of the X-ray dose on room-temperature time-resolved Laue data is discussed.
Protein X-ray structures are determined with ionizing radiation that damages the protein at high X-ray doses. As a result, diffraction patterns deteriorate with the increased absorbed dose. Several strategies such as sample freezing or scavenging of X-ray-generated free radicals are currently employed to minimize this damage. However, little is known about how the absorbed X-ray dose affects time-resolved Laue data collected at physiological temperatures where the protein is fully functional in the crystal, and how the kinetic analysis of such data depends on the absorbed dose. Here, direct evidence for the impact of radiation damage on the function of a protein is presented using time-resolved macromolecular crystallography. The effect of radiation damage on the kinetic analysis of time-resolved X-ray data is also explored.
doi:10.1107/S090904951105549X
PMCID: PMC3284346  PMID: 22338689
radiation damage; X-ray dose; room temperature; time-resolved crystallography; Laue crystallography
10.  Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering 
Nature methods  2008;5(10):881-886.
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.
doi:10.1038/nmeth.1255
PMCID: PMC3159148  PMID: 18806790
11.  Direct Observation of Cooperative Protein Structural Dynamics of Homodimeric Hemoglobin from 100 ps to 10 ms with Pump–Probe X-ray Solution Scattering 
Proteins serve as molecular machines in performing their biological functions, but the detailed structural transitions are difficult to observe in their native aqueous environments in real time. For example, despite extensive studies, the solution-phase structures of the intermediates along the allosteric pathways for the transitions between the relaxed (R) and tense (T) forms have been elusive. In this work, we employed picosecond X-ray solution scattering and novel structural analysis to track the details of the structural dynamics of wild-type homodimeric hemoglobin (HbI) from the clam Scapharca inaequivalvis and its F97Y mutant over a wide time range from 100 ps to 56.2 ms. From kinetic analysis of the measured time-resolved X-ray solution scattering data, we identified three structurally distinct intermediates (I1, I2, and I3) and their kinetic pathways common for both the wild type and the mutant. The data revealed that the singly liganded and unliganded forms of each intermediate share the same structure, providing direct evidence that the ligand photolysis of only a single subunit induces the same structural change as the complete photolysis of both subunits does. In addition, by applying novel structural analysis to the scattering data, we elucidated the detailed structural changes in the protein, including changes in the heme–heme distance, the quaternary rotation angle of subunits, and interfacial water gain/loss. The earliest, R-like I1 intermediate is generated within 100 ps and transforms to the R-like I2 intermediate with a time constant of 3.2 ± 0.2 ns. Subsequently, the late, T-like I3 intermediate is formed via subunit rotation, a decrease in the heme–heme distance, and substantial gain of interfacial water and exhibits ligation-dependent formation kinetics with time constants of 730 ± 120 ns for the fully photolyzed form and 5.6 ± 0.8 μs for the partially photolyzed form. For the mutant, the overall kinetics are accelerated, and the formation of the T-like I3 intermediate involves interfacial water loss (instead of water entry) and lacks the contraction of the heme–heme distance, thus underscoring the dramatic effect of the F97Y mutation. The ability to keep track of the detailed movements of the protein in aqueous solution in real time provides new insights into the protein structural dynamics.
doi:10.1021/ja210856v
PMCID: PMC3337689  PMID: 22494177
12.  Single-crystal Raman spectroscopy and X-ray crystallography at beamline X26-C of the NSLS 
Journal of Synchrotron Radiation  2010;18(Pt 1):37-40.
The collection of absorption and Raman spectroscopic data correlated with X-ray diffraction data allows investigators to understand the atomic structure as well as the electronic and vibrational characteristics of their samples, to identify transiently formed intermediates and to explore mechanistic questions. Raman spectroscopy instrumentation at beamline X26-C at the NSLS is currently available to the general user population.
Three-dimensional structures derived from X-ray diffraction of protein crystals provide a wealth of information. Features and interactions important for the function of macromolecules can be deduced and catalytic mechanisms postulated. Still, many questions can remain, for example regarding metal oxidation states and the interpretation of ‘mystery density’, i.e. ambiguous or unknown features within the electron density maps, especially at ∼2 Å resolutions typical of most macromolecular structures. Beamline X26-C at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (BNL), provides researchers with the opportunity to not only determine the atomic structure of their samples but also to explore the electronic and vibrational characteristics of the sample before, during and after X-ray diffraction data collection. When samples are maintained under cryo-conditions, an opportunity to promote and follow photochemical reactions in situ as a function of X-ray exposure is also provided. Plans are in place to further expand the capabilities at beamline X26-C and to develop beamlines at NSLS-II, currently under construction at BNL, which will provide users access to a wide array of complementary spectroscopic methods in addition to high-quality X-ray diffraction data.
doi:10.1107/S0909049510033601
PMCID: PMC3004251  PMID: 21169688
Raman; single-crystal spectroscopy; X-ray diffraction
13.  3D structures by 2D vibrational spectroscopy 
Accounts of chemical research  2012;45(11):1896-1905.
Conspectus
The development of experiments that can generate molecular movies of changing chemical structures is a major challenge for physical chemistry. But to realize this dream, we not only need to significantly improve existing approaches, but we also must invent new technologies .. Most of the known protein structures have been determined by X-ray diffraction and to lesser extent by NMR. Though powerful, X-ray diffraction presents limitations for acquiring time dependent structures. In the case of NMR, ultrafast equilibrium dynamics might be inferred from lineshapes, but the structures of conformations interconverting on such time scales are not realizable.
This Account highlights two dimensional infrared spectroscopy (2D IR), in particular the 2D vibrational echo, as an approach to time resolved structure determination. We outline the use of the 2D IR method to completely determine the structure of a protein of the integrin family in a time window of few picoseconds. As a transmembrane protein, this class of structures has proved particularly challenging for the established structural methodologies of x-ray crystallography and NMR.
We describe the challenges facing multidimensional spectroscopy and compare it with some other methods of structural biology. Then we succinctly discuss the basic principles of 2D IR methods as they relate to time domain and frequency domain experimental and theoretical properties required for protein structure determination. By means of the example of the transmembrane protein, we describe the essential aspects of combined carbon-13 oxygen-18 isotope labels to create vibrational resonance pairs that allow the determination of protein and peptide structures in motion. Finally, we propose a three dimensional structure of the αIIb transmembrane homodimer that includes optimum locations of all side chains and backbone atoms of the protein.
Delocalization among 13C=18O residues on different helices. The vibrational excitation is transferred between modes on different helices on the coherent energy transfer time π/2β.
doi:10.1021/ar3000025
PMCID: PMC3392492  PMID: 22458539
Multidimensional spectra; vibrational spectra; three dimensional structure; photon echo; vibrational probe; peptide vibrational dynamics
14.  Early Events, Kinetic Intermediates and the Mechanism of Protein Folding in Cytochrome c 
Kinetic studies of the early events in cytochrome c folding are reviewed with a focus on the evidence for folding intermediates on the submillisecond timescale. Evidence from time-resolved absorption, circular dichroism, magnetic circular dichroism, fluorescence energy and electron transfer, small-angle X-ray scattering and amide hydrogen exchange studies on the t ≤ 1 ms timescale reveals a picture of cytochrome c folding that starts with the ~ 1-μs conformational diffusion dynamics of the unfolded chains. A fractional population of the unfolded chains collapses on the 1 – 100 μs timescale to a compact intermediate IC containing some native-like secondary structure. Although the existence and nature of IC as a discrete folding intermediate remains controversial, there is extensive high time-resolution kinetic evidence for the rapid formation of IC as a true intermediate, i.e., a metastable state separated from the unfolded state by a discrete free energy barrier. Final folding to the native state takes place on millisecond and longer timescales, depending on the presence of kinetic traps such as heme misligation and proline mis-isomerization. The high folding rates observed in equilibrium molten globule models suggest that IC may be a productive folding intermediate. Whether it is an obligatory step on the pathway to the high free energy barrier associated with millisecond timescale folding to the native state, however, remains to be determined.
doi:10.3390/ijms10041476
PMCID: PMC2680628  PMID: 19468320
Collapsed intermediate; secondary structure formation; disordered tertiary structure; conformational diffusion; unfolded chains; molten globule; heme misligation; time-resolved spectroscopy; far-UV circular dichroism; magnetic circular dichroism; Trp59 fluorescence; amide hydrogen exchange; small-angle X-ray scattering; thermophiles; three-state pathway
15.  A Single Amino Acid Substitution in the Active Site of Escherichia coli Aspartate Transcarbamoylase Prevents the Allosteric Transition 
Journal of molecular biology  2005;349(2):413-423.
Modeling of the tetrahedral intermediate within the active site of Escherichia coli aspartate transcarbamoylase revealed a specific interaction with the side chain of Gln137, an interaction not previously observed in the structure of the X-ray enzyme in the presence of N-phosphonacetyl-L-aspartate (PALA). Previous site-specific mutagenesis experiments showed that when Gln137 was replaced by alanine, the resulting mutant enzyme (Q137A) exhibited approximately 50-fold less activity than the wild-type enzyme, exhibited no homotropic cooperativity, and the binding of both carbamoyl phosphate and aspartate were extremely compromised. To elucidate the structural alterations in the mutant enzyme that might lead to such pronounced changes in kinetic and binding properties, the Q137A enzyme was studied by time-resolved small-angle X-ray scattering and its structure was determined in the presence of PALA to 2.7Å resolution. Time-resolved small-angle X-ray scattering established that the natural substrates, carbamoyl phosphate and L-aspartate, do not induce in the Q137A enzyme the same conformational changes as observed for the wild-type enzyme, although the scattering pattern of the Q137A and wild-type enzymes in the presence of PALA were identical. The overall structure of the Q137A enzyme is similar to that of the R-state structure of wild-type enzyme with PALA bound. However, there are differences in the manner by which the Q137A enzyme coordinates PALA, especially in the side chain positions of Arg105 and His134. The replacement of Gln137 by Ala also has a dramatic effect on the electrostatics of the active site. These data taken together suggest that the side chain of Gln137 in the wild-type enzyme is required for the binding of carbamoyl phosphate in the proper orientation so as to induce conformational changes required for the creation of the high-affinity aspartate binding site. The inability of carbamoyl phosphate to create the high-affinity binding site in the Q137A enzyme results in an enzyme locked in the low activity low affinity T state. These results emphasize the absolute requirement of the binding of carbamoyl phosphate for the creation of the high-affinity aspartate binding site and for inducing the homotropic cooperativity in aspartate transcarbamoylase.
doi:10.1016/j.jmb.2005.03.073
PMCID: PMC1479453  PMID: 15890205
allosteric enzyme; polar contacts; electrostatics; Poisson-Boltzmann equation; small-angle X-ray scattering; domain closure; allosteric transition; intersubunit interactions
16.  100 ps time-resolved solution scattering utilizing a wide-bandwidth X-ray beam from multilayer optics 
Journal of Synchrotron Radiation  2009;16(Pt 3):391-394.
A new method of time-resolved solution scattering utilizing X-ray multilayer optics is presented.
100 ps time-resolved X-ray solution-scattering capabilities have been developed using multilayer optics at the beamline NW14A, Photon Factory Advanced Ring, KEK. X-ray pulses with an energy bandwidth of ΔE/E = 1–5% are generated by reflecting X-ray pulses (ΔE/E = 15%) through multilayer optics, made of W/B4C or depth-graded Ru/C on silicon substrate. This tailor-made wide-bandwidth X-ray pulse provides high-quality solution-scattering data for obtaining photo-induced molecular reaction dynamics. The time-resolved solution scattering of CH2I2 in methanol is demonstrated as a typical example.
doi:10.1107/S0909049509005986
PMCID: PMC2678014  PMID: 19395804
time-resolved solution scattering; photodissociation reaction; liquidography; multilayers
17.  Sub-100-ps structural dynamics of horse heart myoglobin probed by time-resolved X-ray solution scattering 
Chemical physics  2014;422:137-142.
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. In particular, the global structural change occurring on the time scale of 70 ps is identified.
doi:10.1016/j.chemphys.2014.03.004
PMCID: PMC4323384
Time-resolved X-ray solution scattering; Time-slicing; Structural dynamics; Myoglobin
18.  A 2.8 Å Fe-Fe Separation in the Fe2III/IV Intermediate (X) from Escherichia coli Ribonucleotide Reductase 
Journal of the American Chemical Society  2013;135(45):16758-16761.
A class Ia ribonucleotide reductase (RNR) employs a µ-oxo-Fe2III/III/tyrosyl radical cofactor in its β subunit to oxidize a cysteine residue ~ 35 Å away in its α subunit; the resultant cysteine radical initiates substrate reduction. During self-assembly of the Escherichia coli RNR-β cofactor, reaction of the protein’s Fe2II/II complex with O2 results in accumulation of an Fe2III/IV cluster, termed X, which oxidizes the adjacent tyrosine (Y) 122 to the radical (Y122•) as the cluster is converted to the µ-oxo-Fe2III/III product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class Ia RNRs, X has been the focus of intensive efforts to determine its structure. Initial characterization by extended X-ray absorption fine structure (EXAFS) spectroscopy yielded a 2.5 Å Fe-Fe separation (dFe-Fe), which was interpreted to imply the presence of three single-atom bridges (O2−, HO−, and/or µ-1,1-carboxylates). This short dFe-Fe has been irreconcilable with computational and synthetic models, which all have dFe-Fe ≥ 2.7 Å. To resolve this conundrum, we revisited the EXAFS characterization of X. Assuming that samples containing increased concentrations of the intermediate would yield EXAFS data of improved quality, we applied our recently developed method of generating O2 in situ from chlorite using the enzyme chlorite dismutase to prepare X at ~ 2.0 mM, > 2.5 times the concentration realized in the previous EXAFS study. The measured dFe-Fe of 2.78 Å is fully consistent with computational models containing a (µ-oxo)2-Fe2III/IV core. The correction of dFe–Fe brings the experimental data and computational models into full conformity and thus informs analysis of the mechanism by which X generates Y122•.
doi:10.1021/ja407438p
PMCID: PMC4209742  PMID: 24094084
19.  Structural insights into early folding events using continuous-flow time-resolved SAXS 
Biopolymers  2011;95(8):550-558.
Small-angle x-ray scattering (SAXS) is a powerful method for obtaining quantitative structural information on the size and shape of proteins, and it is increasingly used in kinetic studies of folding and association reactions. In this mini-review, we discuss recent developments in using SAXS to obtain structural information on the unfolded ensemble and early folding intermediates of proteins using continuous-flow mixing devices. Interfacing of these micromachined devices to SAXS beamlines has allowed access to the microsecond time regime. The experimental constraints in implementation of turbulence and laminar flow based mixers with SAXS detection and a comparison of the two approaches are presented. Current improvements and future prospects of microsecond time-resolved SAXS and the synergy with ab initio structure prediction and molecular dynamics simulations are discussed.
doi:10.1002/bip.21628
PMCID: PMC3278217  PMID: 21442608
20.  Five-dimensional crystallography 
Here it is demonstrated how five-dimensional crystallography can be used to determine a comprehensive chemical kinetic mechanism in concert with the atomic structures of transient intermediates that form and decay during the course of the reaction.
A method for determining a comprehensive chemical kinetic mechanism in macromolecular reactions is presented. The method is based on five-dimensional crystallography, where, in addition to space and time, temperature is also taken into consideration and an analysis based on singular value decomposition is applied. First results of such a time-resolved crystallographic study are presented. Temperature-dependent time-resolved X-ray diffraction measurements were conducted on the newly upgraded BioCARS 14-ID-B beamline at the Advanced Photon Source and aimed at elucidating a comprehensive kinetic mechanism of the photoactive yellow protein photocycle. Extensive time series of crystallographic data were collected at two temperatures, 293 K and 303 K. Relaxation times of the reaction extracted from these time series exhibit measurable differences for the two temperatures, hence demonstrating that five-dimensional crystallography is feasible.
doi:10.1107/S0108767309054166
PMCID: PMC2824529  PMID: 20164643
time-resolved crystallography; chemical kinetics; protein structure; temperature dependence
21.  Large crystal growth by thermal control allows combined X-ray and neutron crystallographic studies to elucidate the protonation states in Aspergillus flavus urate oxidase 
Journal of the Royal Society Interface  2009;6(Suppl 5):S599-S610.
Urate oxidase (Uox) catalyses the oxidation of urate to allantoin and is used to reduce toxic urate accumulation during chemotherapy. X-ray structures of Uox with various inhibitors have been determined and yet the detailed catalytic mechanism remains unclear. Neutron crystallography can provide complementary information to that from X-ray studies and allows direct determination of the protonation states of the active-site residues and substrate analogues, provided that large, well-ordered deuterated crystals can be grown. Here, we describe a method and apparatus used to grow large crystals of Uox (Aspergillus flavus) with its substrate analogues 8-azaxanthine and 9-methyl urate, and with the natural substrate urate, in the presence and absence of cyanide. High-resolution X-ray (1.05–1.20 Å) and neutron diffraction data (1.9–2.5 Å) have been collected for the Uox complexes at the European Synchrotron Radiation Facility and the Institut Laue-Langevin, respectively. In addition, room temperature X-ray data were also collected in preparation for joint X-ray and neutron refinement. Preliminary results indicate no major structural differences between crystals grown in H2O and D2O even though the crystallization process is affected. Moreover, initial nuclear scattering density maps reveal the proton positions clearly, eventually providing important information towards unravelling the mechanism of catalysis.
doi:10.1098/rsif.2009.0162.focus
PMCID: PMC2843968  PMID: 19586953
urate oxidase; neutron and X-ray crystallography; crystal growth; phase diagram; H–D exchange; protonation states
22.  Conformational Substates of Myoglobin Intermediate Resolved by Picosecond X-ray Solution Scattering 
Conformational substates of proteins are generally considered to play important roles in regulating protein functions, but an understanding of how they influence the structural dynamics and functions of the proteins has been elusive. Here, we investigate the structural dynamics of sperm whale myoglobin associated with the conformational substates using picosecond X-ray solution scattering. By applying kinetic analysis considering all of the plausible candidate models, we establish a kinetic model for the entire cycle of the protein transition in a wide time range from 100 ps to 10 ms. Four structurally distinct intermediates are formed during the cycle, and most importantly, the transition from the first intermediate to the second one (B → C) occurs biphasically. We attribute the biphasic kinetics to the involvement of two conformational substates of the first intermediate, which are generated by the interplay between the distal histidine and the photodissociated CO.
SECTION
Biophysical Chemistry and Biomolecules
doi:10.1021/jz4027425
PMCID: PMC3985870  PMID: 24761190
23.  Conformational Substates of Myoglobin Intermediate Resolved by Picosecond X-ray Solution Scattering 
Conformational substates of proteins are generally considered to play important roles in regulating protein functions, but an understanding of how they influence the structural dynamics and functions of the proteins has been elusive. Here, we investigate the structural dynamics of sperm whale myoglobin associated with the conformational substates using picosecond X-ray solution scattering. By applying kinetic analysis considering all of the plausible candidate models, we establish a kinetic model for the entire cycle of the protein transition in a wide time range from 100 ps to 10 ms. Four structurally distinct intermediates are formed during the cycle, and most importantly, the transition from the first intermediate to the second one (B → C) occurs biphasically. We attribute the biphasic kinetics to the involvement of two conformational substates of the first intermediate, which are generated by the interplay between the distal histidine and the photodissociated CO.
doi:10.1021/jz4027425
PMCID: PMC3985870  PMID: 24761190
24.  X-Ray Structure determination of the Glycine Cleavage System Protein H of Mycobacterium tuberculosis Using An Inverse Compton Synchrotron X-Ray Source 
Structural genomics discovery projects require ready access to both X-ray and NMR instrumentation which support the collection of experimental data needed to solve large numbers of novel protein structures. The most productive X-ray crystal structure determination laboratories make extensive frequent use of tunable synchrotron X-ray light to solve novel structures by anomalous diffraction methods. This requires that frozen cryo-protected crystals be shipped to large government-run synchrotron facilities for data collection. In an effort to eliminate the need to ship crystals for data collection, we have developed the first laboratory-scale synchrotron light source capable of performing many of the state-of-the-art synchrotron applications in X-ray science. This Compact Light Source is a first-in-class device that uses inverse Compton scattering to generate X-rays of sufficient flux, tunable wavelength and beam size to allow high-resolution X-ray diffraction data collection from protein crystals. We report on benchmarking tests of X-ray diffraction data collection with hen egg white lysozyme, and the successful high-resolution X-ray structure determination of the Glycine cleavage system protein H from Mycobacterium tuberculosis using diffraction data collected with the Compact Light Source X-ray beam.
doi:10.1007/s10969-010-9087-6
PMCID: PMC2939714  PMID: 20364333
25.  Development of a shutterless continuous rotation method using an X-ray CMOS detector for protein crystallography 
Journal of Applied Crystallography  2009;42(Pt 6):1165-1175.
A shutterless continuous rotation method using an X-ray complementary metal-oxide semiconductor (CMOS) detector has been developed for high-speed, precise data collection in protein crystallography. The new method and detector were applied to the structure determination of three proteins by multi- and single-wavelength anomalous diffraction phasing and have thereby been proved to be applicable in protein crystallography.
A new shutterless continuous rotation method using an X-ray complementary metal-oxide semiconductor (CMOS) detector has been developed for high-speed, precise data collection in protein crystallography. The principle of operation and the basic performance of the X-ray CMOS detector (Hamamatsu Photonics KK C10158DK) have been shown to be appropriate to the shutterless continuous rotation method. The data quality of the continuous rotation method is comparable to that of the conventional oscillation method using a CCD detector and, furthermore, the combination with fine ϕ slicing improves the data accuracy without increasing the data-collection time. The new method is more sensitive to diffraction intensity because of the narrow dynamic range of the CMOS detector. However, the strong diffraction spots were found to be precisely measured by recording them on successive multiple images by selecting an adequate rotation step. The new method has been used to successfully determine three protein structures by multi- and single-wavelength anomalous diffraction phasing and has thereby been proved applicable in protein crystallography. The apparatus and method may become a powerful tool at synchrotron protein crystallography beamlines with important potential across a wide range of X-ray wavelengths.
doi:10.1107/S0021889809042277
PMCID: PMC3246825  PMID: 22477775
protein crystallography; shutterless continuous rotation method; X-ray CMOS detectors; X-ray wavelength capabilities

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