Myoglobin Crystals Research Articles

X-ray fluorescence holography of biological metal sites: Application to myoglobin

X-ray fluorescence holography (XFH) is a relatively new technique capable of providing unique three-dimensional structural information around specific atoms that act as a light source in crystalline samples. So far, XFH has typically been applied to inorganic materials such as dopants in metals and semiconductors. Here, we investigate the possibility of using XFH to visualize the metal active site in sperm whale myoglobin (Mb), a monomeric oxygen storage heme protein. We demonstrate that the atomic images reconstructed from the hologram data of crystals of carbonmonoxy myoglobin (MbCO) are moderately consistent with the crystal structure, which is also determined in this study by X-ray crystallography in the near-atomic resolution, as well as simulation results. These results open up a new avenue for the application of XFH to local atomic and electronic structure imaging of metal-sites in biomolecules.

Low-dose fixed-target serial synchrotron crystallography.

The development of serial crystallography has been driven by the sample requirements imposed by X-ray free-electron lasers. Serial techniques are now being exploited at synchrotrons. Using a fixed-target approach to high-throughput serial sampling, it is demonstrated that high-quality data can be collected from myoglobin crystals, allowing room-temperature, low-dose structure determination. The combination of fixed-target arrays and a fast, accurate translation system allows high-throughput serial data collection at high hit rates and with low sample consumption.

Automated grid-based XFEL diffraction studies of single macromolecular crystals

A major challenge of structural investigations of metalloproteins at synchrotrons is the damaging effects of radiation exposure. Even small X-ray doses can reduce or initiate reactions at metal centers modifying the active site. For example, in-situ visible absorption spectroscopy measurements have demonstrated that the heme/copper active site in oxidized ba3 cytochrome oxidase (ba3) is compromised during a single X-ray diffraction exposure. The use of ultrashort X-ray pulses at LCLS provides a means to measure high resolution diffraction before these damage processes occur. To this end, experiments were conducted at LCLS using large multiple crystals (> 50 µm) of ba3, hydrogenase and myoglobin. Crystals were mounted in `grids' or loops and flash frozen. The grids hold up to 75 crystals in known locations and are compatible with the Stanford Automounter used to exchange them. Following a semi-automated grid alignment procedure, a fully automated routine was used to position each crystal and collect a series of diffraction images and the Blu-Ice/DCS control system that coordinated with the LCLS EPICS system and XPP DAQ software. Single femtosecond X-ray pulses produced a `damage free' still diffraction image from each crystal. To provide additional information about crystal orientation, a series of pseudo-oscillation images were collected +/- 5.5 degrees spanning the orientation of the still image. For each one degree oscillation image the crystal was exposed to 120 attenuated X-ray pulses. A hard X-ray spectrometer was used to measure the energy spectrum of each individual X-ray pulse. The details and results of these experiments will be presented.

Radiation Damage of Myoglobin Crystals in Weak Stationary Electric and Magnetic Fields

Radiation damage is one of the bottlenecks in the field of structural biology. Cryo-cooling of protein crystals provided a breakthrough in the 1980s and resulted in significant reductions in radiation damage. Other factors positively influencing the progression of damage include the application of radical scavengers and reductions in the experimental beam size. Here we study the impact on radiation damage of applying static magnetic and electric fields during protein diffraction experiments, ultimately probing the Lorenz force effect on primary photoelectrons and secondary Auger electrons, which both contribute to the damage process. The design of a special mounting pin using graphene for applying electric fields on a crystalline sample is described. Analyses of myoglobin protein crystals exposed to the fields of ~40 mT and -300 V show a slower global radiation damage rate and also changes in the progression of specific damage process on the molecular level, in particular at doses extending beyond the Garman limit of 30 MGy.

Factors correlating with significant differences between X-ray structures of myoglobin.

Validation of general ideas about the origins of conformational differences in proteins is critical in order to arrive at meaningful functional insights. Here, principal component analysis (PCA) and distance difference matrices are used to validate some such ideas about the conformational differences between 291 myoglobin structures from sperm whale, horse and pig. Almost all of the horse and pig structures form compact PCA clusters with only minor coordinate differences and outliers that are easily explained. The 222 whale structures form a few dense clusters with multiple outliers. A few whale outliers with a prominent distortion of the GH loop are very similar to the cluster of horse structures, which all have a similar GH-loop distortion apparently owing to intermolecular crystal lattice hydrogen bonds to the GH loop from residues near the distal histidine His64. The variations of the GH-loop coordinates in the whale structures are likely to be owing to the observed alternative intermolecular crystal lattice bond, with the change to the GH loop distorting bonds correlated with the binding of specific `unusual' ligands. Such an alternative intermolecular bond is not observed in horse myoglobins, obliterating any correlation with the ligands. Intermolecular bonds do not usually cause significant coordinate differences and cannot be validated as their universal cause. Most of the native-like whale myoglobin structure outliers can be correlated with a few specific factors. However, these factors do not always lead to coordinate differences beyond the previously determined uncertainty thresholds. The binding of unusual ligands by myoglobin, leading to crystal-induced distortions, suggests that some of the conformational differences between the apo and holo structures might not be `functionally important' but rather artifacts caused by the binding of `unusual' substrate analogs. The causes of P6 symmetry in myoglobin crystals and the relationship between crystal and solution structures are also discussed.

A history of neutrons in biology: the development of neutron protein crystallography at BNL and LANL

The first neutron diffraction data were collected from crystals of myoglobin almost 42 years ago using a step-scan diffractometer with a single detector. Since then, major advances have been made in neutron sources, instrumentation and data collection and analysis, and in biochemistry. Fundamental discoveries about enzyme mechanisms, biological complex structures, protein hydration and H-atom positions have been and continue to be made using neutron diffraction. The promise of neutrons has not changed since the first crystal diffraction data were collected. Today, with the developments of beamlines at spallation neutron sources and the use of the Laue method for data collection, the field of neutrons in structural biology has renewed vitality.

Tracking ligand-migration pathways of carbonmonoxy myoglobin in crystals at cryogenic temperatures

In order to explore the ligand-migration dynamics in myoglobin induced by photodissociation, cryogenic X-ray crystallographic investigations of carbonmonoxy myoglobin crystals illuminated by continuous wave and pulsed lasers at 1-15 kHz repetition rate have been carried out. Here it is shown that this novel method, extended pulsed-laser pumping of carbonmonoxy myoglobin, promotes ligand migration in the protein matrix by crossing the glass transition temperature repeatedly, and enables the visualization of the migration pathway of the photodissociated ligands in native Mb at cryogenic temperatures. It has revealed that the migration of the CO molecule into each cavity induces structural changes of the amino-acid residues around the cavity which result in the expansion of the cavity. The sequential motion of the ligand and the cavity suggests a self-opening mechanism of the ligand-migration channel arising by induced fit.

Modification of Porous Protein Crystals in Development of Biohybrid Materials

Protein assemblies have attracted increasing attention for construction of biohybrid materials. Protein crystals can also be regarded as solid protein assemblies. The present work demonstrates that protein crystals can be employed as porous biomaterials by site-specific modifications of the crystals of recombinant sperm whale myoglobin mutants. The myoglobin crystals of space group P6 provide hexagonal pores consisting of the building blocks of six Mb molecules, which form a pore with a diameter of 40 A. On the basis of the lattice structure of the Mb crystals, we have selected appropriate residues located on the surface of the pores for replacement with cysteine. This enables modification of the pore surface via coupling with maleimide derivatives. We have succeeded in crystallizing the modified Mb mutants, retaining the P6 lattice, and consistently aligning nanosized functional molecules such as fluorescein, eosin, and Ru(bpy)(3) into the hexagonal pores of the Mb crystals. Our strategy for site-specific modification of protein crystal pores is applicable to various protein crystals with porous structures. We believe that modified porous protein crystals will provide attractive candidates for novel solid materials in nanotechnology applications.

The crystal structure of peroxymyoglobin generated through cryoradiolytic reduction of myoglobin compound III during data collection

Myoglobin has the ability to react with hydrogen peroxide, generating high-valent complexes similar to peroxidases (compounds I and II), and in the presence of excess hydrogen peroxide a third intermediate, compound III, with an oxymyoglobin-type structure is generated from compound II. The compound III is, however, easily one-electron reduced to peroxymyoglobin by synchrotron radiation during crystallographic data collection. We have generated and solved the 1.30 A (1 A=0.1 nm) resolution crystal structure of the peroxymyoglobin intermediate, which is isoelectric to compound 0 and has a Fe-O distance of 1.8 A and O-O bond of 1.3 A in accordance with a Fe(II)-O-O- (or Fe(III)-O-O2-) structure. The generation of the peroxy intermediate through reduction of compound III by X-rays shows the importance of using single-crystal microspectrophotometry when doing crystallography on metalloproteins. After having collected crystallographic data on a peroxy-generated myoglobin crystal, we were able (by a short annealing) to break the O-O bond leading to formation of compound II. These results indicate that the cryoradiolytic-generated peroxymyoglobin is biologically relevant through its conversion into compound II upon heating. Additionally, we have observed that the Xe1 site is occupied by a water molecule, which might be the leaving group in the compound II to compound III reaction.

A new rotation function for molecular replacement by using both the self and cross Patterson vectors

The rotation function in molecular replacement is traditionally computed in reciprocal space. The common practice is to use a Patterson vector radius of about the size of the target molecule to limit the interatomic vectors to match only the Patterson self-vectors. In real space, the molecular image of a search model can be searched and matched to the Patterson function vector space. Depending on which asymmetric unit the matched Patterson vectors are in, both self or cross Patterson vectors can be matched to the search model. The algorithm described here for computing the rotation function is to average the signals from all images found by the image-seeking functions in the Patterson vector search. Tests show that a search model consisting of a two-helix fragment can be found in a myoglobin crystal using experimental data in a global rotation search. This demonstrates that the new rotation function is a potentially useful approach.

S-Nitrosylation-induced Conformational Change in Blackfin Tuna Myoglobin

S-nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by "wedging" apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-A structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket.

X-ray structure analysis of a metalloprotein with enhanced active-site resolution using in situ x-ray absorption near edge structure spectroscopy

X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbing atom and therefore allows bond distances and angles of the surrounding atomic cluster to be measured with atomic resolution. By contrast, the accuracy and resolution of metalloprotein active sites obtainable from x-ray crystallography are often insufficient to analyze the electronic properties of the metals that are essential for their biological functions. Here, we demonstrate that the combination of both methods on the same metalloprotein single crystal yields a structural model of the protein with exceptional active-site resolution. To this end, we have collected an x-ray diffraction data set to 1.4-A resolution and Fe K-edge polarized x-ray absorption near edge structure (XANES) spectra on the same cyanomet sperm whale myoglobin crystal. The XANES spectra were quantitatively analyzed by using a method based on the multiple scattering approach, which yielded Fe-heme structural parameters with +/-(0.02-0.07)-A accuracy on the atomic distances and +/-7 degrees on the Fe-CN angle. These XANES-derived parameters were subsequently used as restraints in the crystal structure refinement. By combining XANES and x-ray diffraction, we have obtained an cyanomet sperm whale myoglobin structural model with a higher precision of the bond lengths and angles at the active site than would have been possible with crystallographic analysis alone.

Cryoradiolytic reduction of crystalline heme proteins: analysis by UV-Vis spectroscopy and X-ray crystallography

The X-ray crystallographic analysis of redox-active systems may be complicated by photoreduction. Although radiolytic reduction by the probing X-ray beam may be exploited to generate otherwise short-lived reaction intermediates of metalloproteins, it is generally an undesired feature. Here, the X-ray-induced reduction of the three heme proteins myoglobin, cytochrome P450cam and chloroperoxidase has been followed by on-line UV-Vis absorption spectroscopy. All three systems showed a very rapid reduction of the heme iron. In chloroperoxidase the change of the ionization state from ferric to ferrous heme is associated with a movement of the heme-coordinating water molecule. The influence of the energy of the incident X-ray photons and of the presence of scavengers on the apparent reduction rate of ferric myoglobin crystals was analyzed.

Crystal Structure and Peroxidase Activity of Myoglobin Reconstituted with Iron Porphycene

The incorporation of an artificially created metal complex into an apomyoglobin is one of the attractive methods in a series of hemoprotein modifications. Single crystals of sperm whale myoglobin reconstituted with 13,16-dicarboxyethyl-2,7-diethyl-3,6,12,17-tetramethylporphycenatoiron(III) were obtained in the imidazole buffer, and the 3D structure with a 2.25-A resolution indicates that the iron porphycene, a structural isomer of hemin, is located in the normal position of the heme pocket. Furthermore, it was found that the reconstituted myoglobin catalyzed the H2O2-dependent oxidations of substrates such as guaiacol, thioanisole, and styrene. At pH 7.0 and 20 degrees C, the initial rate of the guaiacol oxidation is 11-fold faster than that observed for the native myoglobin. Moreover, the stopped-flow analysis of the reaction of the reconstituted protein with H2O2 suggested the formation of two reaction intermediates, compounds II- and III-like species, in the absence of a substrate. It is a rare example that compound III is formed via compound II in myoglobin chemistry. The enhancement of the peroxidase activity and the formation of the stable compound III in myoglobin with iron porphycene mainly arise from the strong coordination of the Fe-His93 bond.

Coherent infrared emission from myoglobin crystals: an electric field measurement.

We introduce coherent infrared emission interferometry as a chi(2) vibrational spectroscopy technique and apply it to studying the initial dynamics upon photoactivation of myoglobin (Mb). By impulsive excitation (using 11-fs pulses) of a Mb crystal, vibrations that couple to the optical excitation are set in motion coherently. Because of the order in the crystal lattice the coherent oscillations of the different proteins in the crystal that are associated with charge motions give rise to a macroscopic burst of directional multi-teraHertz radiation. This radiation can be detected in a phase-sensitive way by heterodyning with a broad-band reference field. In this way both amplitude and phase of the different vibrations can be obtained. We detected radiation in the 1,000-1,500 cm(-1) frequency region, which contains modes sensitive to the structure of the heme macrocycle, as well as peripheral protein modes. Both in carbonmonoxy-Mb and aquomet-Mb we observed emission from six modes, which were assigned to heme vibrations. The phase factors of the modes contributing to the protein electric field show a remarkable consistency, taking on values that indicate that the dipoles are created "emitting" at t = 0, as one would expect for impulsively activated modes. The few deviations from this behavior in Mb-CO we propose are the result of these modes being sensitive to the photodissociation process and severely disrupted by it.

Multiple CCD detector for macromolecular X-ray crystallography

A charge-coupled device (CCD)-based detector designed for macromolecular crystallography is described. The detector has an area of 200 × 200 mm, a readout time of 1.6 s, and total noise equivalent to approximately three 12 keV X-ray photons per pixel. The detector is constructed from a 2 × 2 array of four identical units, each unit consisting of a 4.1:1 demagnifying fiber-optic taper bonded to a 1 k × 1 k, 24 µm pixel, CCD sensor. Each CCD is read out in parallel though four channels and digitized to 16 bits. A Gd2O2S phosphor X-ray-to-light converter bonded to an aluminized-plastic film is held in contact with the input surfaces of the fiber-optic tapers with an air pillow. The full width at half-maximum (FWHM) of the point response function is 120 µm, the response is linear to better than 1% over the entire range of intensity from background to nearly full well, the gain is 3.4 e per 8 keV incident X-ray photon, the noise is 12.6 e per pixel for a 10 s integration time, the modulation transfer function (MTF) is 0.35 at 5 line pairs (lp) mm−1(the Nyquest frequency), and the measured detective quantum efficiency (DQE) is 0.74 for relatively strong Bragg peaks. Data collected from crystallographic studies with synchrotron radiation are presented. In an anomalous difference Patterson map for a data set collected in 40 min on a monoclinic myoglobin crystal, the magnitude of the Fe–Fe peaks is 18 times the standard uncertainty of the map.

Angular- and temperature-dependent RSMR on myoglobin using 183 W and 57 Fe

Myoglobin crystals are investigated by Rayleigh scattering of Mossbauer radiation at T = 87 K and T = 300 K and angles up to sin(ϑ)/λ = 0.44 A−1. The results are analysed in terms of normal modes.

Use of an open-flow helium cryostat for macromolecular cryocrystallography

A helium cryostat developed at the University of Toledo has recently been described by Hardie, Kirschbaum, Martin & Pinkerton [J. Appl. Cryst.(1998),31, 815–817]. This helium cooling system has now been tested on macromolecules, using crystals of chicken egg lysozyme and sperm whale myoglobin. Phase changes in terbium vanadate crystals indicate that temperatures delivered by the cryostat were less than 33 K. Unit-cell contraction in the protein crystals is consistent with the previously reported He data. Large crystals approaching suitability for neutron diffraction studies were successfully flash-cooled and a large crystal of lysozyme was rescued for data collection after undergoing a cycle of macromolecular crystal annealing (MCA) following poor flash-cooling. Comparison of diffraction data from sperm whale myoglobin crystals collected in both He and N2showed a 23% lower overallBfactor for the He data. The results of these studies show that this device might be an especially useful adjunct both for neutron diffraction studies and at high-intensity synchrotron X-ray sources, in addition to its use in the standard macromolecular crystallography laboratory.

A low-resolution low-temperature neutron diffraction study of myoglobin.

Diffraction data to 5 A resolution were collected on a myoglobin crystal at 80, 130, 180 and 240 K. The linear coefficient of thermal expansion for myoglobin was determined to be 45 x 10(-6) K(-1), based on the measured expansion of the unit-cell parameters. The nature of the hydration layers surrounding the protein in the crystal is described in terms of a shell solvent model, which was used to calculate the coefficient of thermal expansion in reasonable agreement with the measured value. Wilson statistics were calculated and discussed in terms of an averaged disorder model. [F(T(2)) - F(80 K) exp(-iphi)] Fourier maps were calculated where T(2) was taken as 130, 180 and 240 K, respectively. None of these difference maps showed any features above 2.0sigma in the protein region. The 130 and 240 K difference maps showed many small and widely distributed negative difference features and showed very few positive difference features above 2.5sigma in the solvent region. However, the 180 K difference map showed an extensive negative difference feature at the interface between symmetry-related molecules, occurring in the vicinity of residues 40-50 on one molecule and 76-80 on a symmetry-related molecule. These difference neutron Fourier maps indicate a concerted effect at 180 K, which is interpreted in terms of an onset of extended lattice disorder.

Successful flash-cooling of xenon-derivatized myoglobin crystals

This paper demonstrates for the first time a method for preparing cryocooled xenon-derivatized protein crystals. The method is based upon the hypothesis and subsequent observation that the diffusion of a xenon atom from a tight binding site following depressurization occurs on a timescale of minutes. We have observed significant changes in diffraction intensities from myoglobin crystals for up to 5 min following depressurization from 1 MPa of xenon. In accordance with this observation, a xenon-derivatized myoglobin crystal was cryocooled at ~95 K within 20 s of complete depressurization. A crystallographic data set was then collected to 2.0 A resolution and isomorphous and anomalous difference Patterson maps revealed the presence of a well ordered xenon site with an occupancy of approximately 0.5. Phasing statistics for this site were of good quality and demonstrate the practicality of this method. The ability to cryocool xenon-derivatized crystals will make this heavy-atom substitution method even more useful for single-isomorphous-replacement and multiple-isomorphous-replacement phasing of macromolecules.