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.
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.
X-ray free-electron lasers; structural biology; serial femtosecond crystallography; time-resolved diffraction; time-resolved wide angle X-ray scattering
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.
photoisomerization; signal transduction; time-resolved small-angle x-ray scattering; photoactive yellow protein
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.
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.
electron crystallography; electron diffraction; electron cryomicroscopy (cryo-EM); microED; protein structure; microcrystals; None
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.
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.
FTIR spectroscopy; ligand binding; myoglobin; temperature derivative spectroscopy
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.
RNA crystallography; ribozyme; crystallization; RNA structure; crystallographic ensembles; alternate conformation; long-range motion; fold and function; difference Fourier; non-protein-coding RNA
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.
radiation damage; X-ray dose; room temperature; time-resolved crystallography; Laue crystallography
Proteins serve as molecular machines in performing their
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.
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.
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
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.
allosteric enzyme; polar contacts; electrostatics; Poisson-Boltzmann equation; small-angle X-ray scattering; domain closure; allosteric transition; intersubunit interactions
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.
Multidimensional spectra; vibrational spectra; three dimensional structure; photon echo; vibrational probe; peptide vibrational dynamics
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.
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
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.
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.
Raman; single-crystal spectroscopy; X-ray diffraction
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.
time-resolved crystallography; chemical kinetics; protein structure; temperature dependence
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•.
In kinetic crystallography, the usually static method of X-ray diffraction is expanded to allow time-resolved analysis of conformational rearrangements in protein structures. To achieve this, reactions have to be triggered within the protein crystals of interest, and optical spectroscopy can be used to monitor the reaction state. For this approach, a modified form of H-Ras p21 was designed which allows reaction initiation and fluorescence readout of the initiated GTPase reaction within the crystalline state. Rearrangements within the crystallized protein due to the progressing reaction and associated heterogeneity in the protein conformations have to be considered in the subsequent refinement processes.
X-ray diffraction experiments on H-Ras p21 in different states along the reaction pathway provide detailed information about the kinetics and mechanism of the GTPase reaction. In addition, a very high data quality of up to 1.0 Å resolution allowed distinguishing two discrete subconformations of H-Ras p21, expanding the knowledge about the intrinsic flexibility of Ras-like proteins, which is important for their function. In a complex of H-Ras•GppNHp (guanosine-5'-(β,γ-imido)-triphosphate), a second Mg2+ ion was found to be coordinated to the γ-phosphate group of GppNHp, which positions the hydrolytically active water molecule very close to the attacked γ-phosphorous atom.
For the structural analysis of very high-resolution data we have used a new 'two-chain-isotropic-refinement' strategy. This refinement provides an alternative and easy to interpret strategy to reflect the conformational variability within crystal structures of biological macromolecules. The presented fluorescent form of H-Ras p21 will be advantageous for fluorescence studies on H-Ras p21 in which the use of fluorescent nucleotides is not feasible.
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.
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.
urate oxidase; neutron and X-ray crystallography; crystal growth; phase diagram; H–D exchange; protonation states
Protein kinases use ATP as a phosphoryl donor for the posttranslational modification of signaling targets. It is generally thought that the binding of this nucleotide induces conformational changes leading to closed, more compact forms of the kinase domain that ideally orient active-site residues for efficient catalysis. The kinase domain is oftentimes flanked by additional ligand binding domains that up- or down-regulate catalytic function. C-terminal Src kinase (Csk) is a multidomain tyrosine kinase that is up-regulated by N-terminal SH2 and SH3 domains. Although the X-ray structure of Csk suggests the enzyme is compact, X-ray scattering studies indicate that the enzyme possesses both compact and open conformational forms in solution. Here, we investigated whether interactions with the ATP analog AMP-PNP and ADP can shift the conformational ensemble of Csk in solution using a combination of small angle x-ray scattering and molecular dynamics simulations. We find that binding of AMP-PNP shifts the ensemble towards more extended rather than more compact conformations. Binding of ADP further shifts the ensemble towards extended conformations, including highly extended conformations not adopted by the apo protein, nor by the AMP-PNP bound protein. These ensembles indicate that any compaction of the kinase domain induced by nucleotide binding does not extend to the overall multi-domain architecture. Instead, assembly of an ATP-bound kinase domain generates further extended forms of Csk that may have relevance for kinase scaffolding and Src regulation in the cell.
The Src protein kinases are integral members of numerous signaling pathways involved in cellular growth and differentiation. The master regulator of the Src family is the protein kinase Csk, which adds a phosphate to the C-terminal tail, inhibiting Src Kinase function. Proper regulation of these signaling pathways by Csk is essential as unregulated activity in these pathways is correlated with the development of various cancers and autoimmune diseases. Understanding the nature of the mechanism and structure of Csk may lead to therapeutics and a better understanding of Src signaling pathways. Conformational changes associated with nucleotide binding and release have been shown to regulate the efficiency of Src down-regulation by Csk. To obtain insights into the nature of these nucleotide-induced structural changes, we examined the conformation of Csk in solution while bound to the ATP analog AMP-PNP and product ADP using a combination of small angle x-ray scattering and molecular dynamics. Surprisingly, both nucleotides induce extended conformations of Csk compared to the apo-enzyme, suggesting a novel mode of function. Further understanding of this mode of function may aid in the design of cancer therapeutics that act by regulating Src signaling pathways by modulating the function of Csk.
Third generation synchrotron sources such as the Advanced Photon Source at Argonne National Laboratory, Argonne, IL, are outstanding tools for X-ray diffraction and scattering studies of non-crystalline biological materials. However, these studies are hindered by the lack of adequate detectors that can provide multiple frames of detailed structural information on the required millisecond time scale at the extremely high count rates available at the APS. RMD is developing a cost effective detector for time-resolved small angle X-ray scattering, using a cooled, 512×512 pixel electron multiplying CCD (EMCCD). This paper describes the detector design, its efficacy for time-resolved SAXS studies, and its imaging performance with frame rates of 30 to 500 fps.
Synchrotron detector; EMCCD; SAXS; time resolved SAXS; Advanced Photon Source
The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals has been explored.
The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using β2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-ray minibeam. SHG imaging was found to provide about 2 µm spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed.
second-harmonic generation microscopy; crystal centering; imaging
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.
Time-resolved X-ray solution scattering; Time-slicing; Structural dynamics; Myoglobin
The L to M reaction of the bacteriorhodopsin photocycle includes the crucial proton transfer from the retinal Schiff base to Asp85. In spite of the importance of the L state in deciding central issues of the transport mechanism in this pump, the serious disagreements among the three published crystallographic structures of L have remained unresolved. Here we report on the x-ray diffraction structure of the L state, to 1.53 – 1.73 Å resolutions, from replicate data sets collected from six independent crystals. Unlike in earlier studies, the partial occupancy refinement uses diffraction intensities from the same crystals before and after the illumination to produce the trapped L state. The high reproducibility of inter-atomic distances, and bond angles and torsions of the retinal, lends credibility to the structural model. The photoisomerized 13-cis retinal in L is twisted at the C13=C14 and C15=NZ double-bonds so that the Schiff base does not lose its connection to wat402 and therefore to the proton acceptor Asp85. The protonation of Asp85 by the Schiff base in the L → M reaction is likely to occur, therefore, via wat402. It is evident from the L state structure that various conformational changes involving hydrogen-bonding residues and bound water begin to propagate from the retinal to the protein at this stage already, and in both extracellular and cytoplasmic directions. Their rationales in the transport can be deduced from the way their amplitudes increase in the intermediates that follow L in the reaction cycle, and from the proton transfer reactions with which they are associated.
bacteriorhodopsin; proton pumps; transport mechanism; membrane proteins; protein crystallography; photointermediates