Multiwavelength anomalous diffraction analysis at the M absorption edges of uranium

The multiwavelength anomalous diffraction (MAD) method for phase evaluation is now widely used in macromolecular crystallography. Successful MAD structure determinations have been carried out at the K or L absorption edges of a variety of elements. In this study, we investigate the anomalous scattering properties of uranium at its M(IV) (3.326 A) and M(V) (3.490 A) edge. Fluorescence spectra showed remarkably strong anomalous scattering at these edges (f' = -70e, f" = 80e at the M(IV) edge and f' = -90e, f" = 105e at the M(V) edge), many times higher than from any anomalous scatterers used previously for MAD phasing. However, the large scattering angles and high absorption at the low energies of these edges present some difficulties not found in typical crystallographic studies. We conducted test experiments at the M(IV) edge with crystals of porcine elastase derivatized with uranyl nitrate. A four-wavelength MAD data set complete to 3.2-A Bragg spacings was collected from a single small frozen crystal. Analysis of the data yielded satisfactory phase information (average difference of (0)phi(T) - (0)phi(A) for replicated determinations is 32 degrees ) and produced an interpretable electron-density map. Our results demonstrate that it is practical to measure macromolecular diffraction data at these edges with current instrumentation and that phase information of good accuracy can be extracted from such experiments. We show that such experiments have potential for the phasing of very large macromolecular assemblages.     

X-ray absorption spectroscopy using synchrotron radiation for structural investigation of organometallic molecules of biological interest.

The technique of x-ray absorption spectroscopy using tuneable, very intense x-rays from a high energy electron storage ring has been applied to study of the estended x-ray absorption fine structure for Cu and Ni tetraphenylporphyrin and methemoglobin. Preliminary analysis shows that the spectra may be interpreted as a super-position of modulations arising from the nearest neighbor nitrogen and pyrrole alpha-carbon coordination sheels of the metal atoms. We estimate that with the observed magnitude of noise to modulation amplitude, relative shifts of 0,5% in the metal-nitrogen to metal-carbon bond distances in the prophyrins should be observable using extended x-ray absorption fine structure and that this technique may provide a method of observing these types of structural changes in solution.     

Tetrahedral structure or chains for liquid water

It has been suggested, based on x-ray absorption spectroscopy (XAS) experiments on liquid water [Wernet, Ph., et al. (2004) Science 304, 995-999], that a condensed-phase water molecule's asymmetric electron density results in only two hydrogen bonds per water molecule on average. The larger implication of the XAS interpretation is that the conventional view of liquid water being a tetrahedrally coordinated random network is now replaced by a structural organization that instead strongly favors hydrogen-bonded water chains or large rings embedded in a weakly hydrogen-bonded disordered network. This work reports that the asymmetry of the hydrogen density exhibited in the XAS experiments agrees with reported x-ray scattering structure factors and intensities for Q > 6.5 A(-1). However, the assumption that the asymmetry in the hydrogen electron density does not fluctuate and is persistent in all local molecular liquid water environments is inconsistent with longer-ranged tetrahedral network signatures present in experimental x-ray scattering intensity and structure factor data for Q < 6.5 A(-1).     

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.     

Circular differential scattering can be an important part of the circular dichroism of macromolecules.

Differential scattering of incident left and right circularly polarized light can be an important contribution to the circular dichroism of macromolecules. In principle both differential absorption and differential scattering of circularly polarized light contribute to circular dichroism, but differential scattering is increasingly important for particles whose dimensions are greater than 1/20th the wavelength of light. The scattering contribution is probably not important for unaggregated proteins and nucleic acids in solution. It can be very important for viruses, membranes, and other protein-nucleic acid complexes. Outside the absorption bands of the scattering, chiral particle, only differential scattering contributes to the circular dichroism. The sign and magnitude of the differential scattering is quantitatively related to the relative orientations and the distances between the scattering units of the particle. The interpretation of the circular differential scattering depends on a simple, classical method. Thus, in understanding a measured circular dichroism, it often will be easier to relate the differential scattering to the structure of a particle (such as a virus) than it is to relate the differential absorption to the structure.     

X-ray damage to the Mn4Ca complex in single crystals of photosystem II: a case study for metalloprotein crystallography

X-ray absorption spectroscopy was used to measure the damage caused by exposure to x-rays to the Mn(4)Ca active site in single crystals of photosystem II as a function of dose and energy of x-rays, temperature, and time. These studies reveal that the conditions used for structure determination by x-ray crystallography cause serious damage specifically to the metal-site structure. The x-ray absorption spectra show that the structure changes from one that is characteristic of a high-valent Mn(4)(III(2),IV(2)) oxo-bridged Mn(4)Ca cluster to that of Mn(II) in aqueous solution. This damage to the metal site occurs at a dose that is more than one order of magnitude lower than the dose that results in loss of diffractivity and is commonly considered safe for protein crystallography. These results establish quantitative x-ray dose parameters that are applicable to redox-active metalloproteins. This case study shows that a careful evaluation of the structural intactness of the active site(s) by spectroscopic techniques can validate structures derived from crystallography and that it can be a valuable complementary method before structure-function correlations of metalloproteins can be made on the basis of high-resolution x-ray crystal structures.     

Applications of synchrotron radiation to protein crystallography: preliminary results.

X-ray diffraction photographs of protein single crystals have been obtained using synchrotron radiation produced by an electron-positron storage ring. The diffracted intensities observed with this unconventional source are a factor of at least 60 greater than those obtained with a sealed x-ray tube using the same crystal and instrumental parameters. Diffraction data have been collected by the precession method to higher resolution and using smaller protein crystals than would have been possible with a conventional source. The crystal decay rate in the synchrotron beam for several proteins appears to be substantially less than that observed with Ni-filtered Cu radiation. The tunable nature of the source (which allows selective optimization of anomalous contributions to the scattering factors) and the low angular divergence of the beam make the source very useful for single crystal protein diffraction studies.     

Preparation and properties of nickel hemoglobin

Hemoglobin A reconstituted with nickel protoporphyrin IX (NiHbA) has been prepared and characterized. Kinetics of its reaction with p-mercuribenzoate and with haptoglobin, absorption and circular dichroism spectra, and x-ray crystallographic properties have been investigated as probes of its structural conformation. The results suggest that NiHbA exists in a structure that is similar to the deoxy, or T-state of HbA. It is proposed that NiHbA and its derivatives may serve as a useful model for future studies of hemoglobin allosteric changes.     

Interpreting hemoglobin and water concentration, oxygen saturation, and scattering measured in vivo by near-infrared breast tomography

Near-infrared spectroscopic tomography was used to measure the properties of 24 mammographically normal breasts to quantify whole-breast absorption and scattering spectra and to evaluate which tissue composition characteristics can be determined from these spectra. The absorption spectrum of breast tissue allows quantification of (i) total hemoglobin concentration, (ii) hemoglobin oxygen saturation, and (iii) water concentration, whereas the scattering spectrum provides information about the size and number density of cellular components and structural matrix elements. These property data were tested for correlation to demographic information, including subject age, body mass index, breast size, and radiographic density. Total hemoglobin concentration correlated inversely to body mass index, likely because lower body mass indicates proportionately less fat and more glandular tissue, and glandular tissue contains greater vascularity, hence, more total hemoglobin. Optical scattering was correlated to breast diameter, subject age, and radiographic density. In the radiographic density, fatty breasts had low scattering power and extremely dense breasts had higher values. This observation is consistent with low attenuation of conventional x-rays with fat and higher attenuation in glandular tissues. Optically, fatty tissues have large scatterers leading to a low scattering power, whereas glandular or fibrous tissues have more cellular and collagen-based structures that lead to high scattering power. The study presents correlative data supporting the hypothesis that optical measurements of absorption and scattering can provide physiologically relevant information about breast tissue composition. These breast constituents vary significantly between individuals and can be altered because of changes in breast physiology or pathological state.     

Rapid compaction during RNA folding

We have used small angle x-ray scattering and computer simulations with a coarse-grained model to provide a time-resolved picture of the global folding process of the Tetrahymena group I RNA over a time window of more than five orders of magnitude. A substantial phase of compaction is observed on the low millisecond timescale, and the overall compaction and global shape changes are largely complete within one second, earlier than any known tertiary contacts are formed. This finding indicates that the RNA forms a nonspecifically collapsed intermediate and then searches for its tertiary contacts within a highly restricted subset of conformational space. The collapsed intermediate early in folding of this RNA is grossly akin to molten globule intermediates in protein folding.     

Magnetars

Recent x-ray observations have shown that a substantial fraction of newly born neutron stars have magnetic fields of several 10(14) G. They reveal themselves as soft gamma repeaters and anomalous x-ray pulsars and may account for the missing radio pulsars in young supernova remnants.     

Functional role of internal water molecules in rhodopsin revealed by X-ray crystallography

Activation of G protein-coupled receptors (GPCRs) is triggered and regulated by structural rearrangement of the transmembrane heptahelical bundle containing a number of highly conserved residues. In rhodopsin, a prototypical GPCR, the helical bundle accommodates an intrinsic inverse-agonist 11-cis-retinal, which undergoes photo-isomerization to the all-trans form upon light absorption. Such a trigger by the chromophore corresponds to binding of a diffusible ligand to other GPCRs. Here we have explored the functional role of water molecules in the transmembrane region of bovine rhodopsin by using x-ray diffraction to 2.6 A. The structural model suggests that water molecules, which were observed in the vicinity of highly conserved residues and in the retinal pocket, regulate the activity of rhodopsin-like GPCRs and spectral tuning in visual pigments, respectively. To confirm the physiological relevance of the structural findings, we conducted single-crystal microspectrophotometry on rhodopsin packed in our three-dimensional crystals and show that its spectroscopic properties are similar to those previously found by using bovine rhodopsin in suspension or membrane environment.     

Oxidants, ATP depletion, and endothelial permeability to macromolecules.

Oxidants can reversibly increase the permeability of endothelium to ions and macromolecules. Oxidants also deplete ATP in cultured endothelial cells. We asked if oxidant-mediated ATP depletion, alone, accounted for the effects of oxidants on endothelial permeability to macromolecules. When porcine pulmonary artery endothelial cells were exposed to 2.5 mmol/LH2O2, ATP was depleted to 31.7% +/- 1.8% of control within 15 minutes and was reduced to 23.1% +/- 2.0% of control after 30 minutes. To determine if this magnitude of ATP depletion could account for the oxidant-induced increase in endothelial permeability to macromolecules, we measured ATP in endothelial cells exposed to metabolic inhibitors of ATP production. We then measured the effects of these metabolic inhibitors on endothelial monolayer permeability to macromolecules. ATP levels were reduced to 44% +/- 4% of control by 12 mmol/L deoxyglucose (DOG) in the absence of glucose and to 2% +/- 1.3% of control by DOG with 25 nmol/L antimycin A in the absence of glucose. Reduction of endothelial cell ATP to these levels with the metabolic inhibitors did not alter the flux of albumin or dextran across the endothelial monolayers. Thus ATP depletion, by itself, does not explain oxidant-induced changes in endothelial permeability to macromolecules.     

Structure of human plasma low-density lipoproteins: molecular organization of the central core.

Human plasma low density lipoprotein (LDL) exhibits a thermal transition over the temperature range 20-40 degrees. This transition is associated with a structural change within the lipoprotein particle and is reflected in the small-angle x-ray scattering profiles from LDL. The scattering profile of the quasispherical LDL particle at 10 degrees shows a relatively intense maximum at 1/36 A-1 which is absent from the scattering of LDL at 45 degrees. Theoretical calculations, using model electron density distributions, have been carried out to describe the packing of arrangement of the cholesterol esters, based on perturbations of the molecular packing of crystalline cholesteryl myristate, adequately reproduces the high relative intensity of the x-ray scattering maximum at 1/36 A-1. The perturbations of the packing in the crystal structure of cholesteryl myristate involve "melting" of the hydrocarbon chains of the esters together with translations of pairs of molecules parallel to the molecular long axis. The interaction of opposing steroid moieties, with C18 and C19 angular methyl groups interlocked, exhibited in the crystal structure is retained in the perturbed arrangement. At 45 degrees, thermally induced disorder of this arrangement averages the electron density of the central core. The x-ray scattering profiles of particles with a homogeneous electron density in the core region do not show a high relative intensity of the subsidiary maxima in the 1/36 A-1 region, in agreement with experimental observation. The results of these calculations support the concept that the thermal transition observed for LDL is due to a smectic leads to disordered transition of the cholesterol esters in the core of the LDL particle.     

Allostery of actin filaments: molecular dynamics simulations and coarse-grained analysis

The structural and mechanical properties of monomeric actin (G-actin), the trimer nucleus, and actin filaments (F-actins) are determined as a function of the conformation of the DNase I-binding loop (DB loop) by using all-atom molecular dynamics simulations and coarse-grained (CG) analysis. Recent x-ray structures of ADP-bound G-actin (G-ADP) by Otterbein et al. [Otterbein, L. R., Graceffa, P. & Dominguez, R. (2001) Science 293, 708-711] and ATP-bound G-actin (G-ATP) by Graceffa and Dominguez [Graceffa, P. & Dominguez, R. (2003) J. Biol. Chem. 278, 34172-34180] indicate that the DB loop of actin does not have a well defined secondary structure in the ATP state but folds into an alpha-helix in the ADP state. MD simulations and CG analysis indicate that such a helical DB loop significantly weakens the intermonomer interactions of actin assemblies and thus leads to a wider, shorter, and more disordered filament. The computed persistence lengths of F-actin composed of G-ATP (16 microm) and of G-ADP (8.5 microm) agree well with the experimental values for the two states. Therefore, the loop-to-helix transition of the DB loop may be one of the factors that lead to the changes in structural and mechanical properties of F-actin after ATP hydrolysis. This result may provide a direct connection between the conformational changes of an actin monomer and the structural and mechanical properties of the cytoskeleton. The information provided by MD simulations also helps to understand the possible origin of the special features of actin dynamics.     

Femtosecond time resolution in x-ray diffraction experiments

This paper presents the theoretical background for a synthesis of femtosecond spectroscopy and x-ray diffraction. When a diffraction quality crystal with 0.1–0.3 mm overall dimensions is photoactivated by a femtosecond laser pulse (physical length = 0.3 μm), the evolution of molecules at separated points in the crystal will not be simultaneous because a finite time is required for the laser pulse to propagate through the body of the crystal. Utilizing this lack of global crystal synchronization, topographic x-ray diffraction may enable femtosecond temporal resolution to be achieved from reflection profiles in the diffraction pattern with x-ray exposures of picosecond or longer duration. Such x-ray pulses are currently available, and could be used to study femtosecond reaction dynamics at atomic resolution on crystals of both small- and macromolecules. A general treatment of excitation and diffraction geometries in relation to spatial and temporal resolution is presented.     

Extended x-ray absorption fine structure studies of a retrovirus: equine infectious anemia virus cysteine arrays are coordinated to zinc.

Zinc finger arrays have been established as a critical structural feature of proteins involved in DNA recognition. Retroviral nucleocapsid proteins, which are involved in the binding of viral RNA, contain conserved cysteine-rich arrays that have been suggested to coordinate zinc. We provide metalloprotein structural data from an intact virus preparation that validate this hypothesis. Extended x-ray absorption fine structure (EXAFS) spectroscopy of well-characterized and active preparations of equine infectious anemia virus, compared with a peptide with known coordination and in combination with available biochemical and genetic data, defines a Cys3His1 coordination environment for zinc. The average of the Zn-S distances is 2.30(1) A and that of the Zn-N distance (to histidine) is 2.01(3) A.     

Cartilage in normal and osteoarthritis conditions

The preservation of articular cartilage depends on keeping the cartilage architecture intact. Cartilage strength and function depend on both the properties of the tissue and on their structural parameters. The main structural macromolecules are collagen and proteoglycans (aggrecan). During life, cartilage matrix turnover is mediated by a multitude of complex autocrine and paracrine anabolic and catabolic factors. These act on the chondrocytes and can lead to repair, remodeling or catabolic processes like those that occur in osteoarthritis. Osteoarthritis is characterized by degradation and loss of articular cartilage, subchondral bone remodeling, and, at the clinical stage of the disease, inflammation of the synovial membrane. The alterations in osteoarthritic cartilage are numerous and involve morphologic and metabolic changes in chondrocytes, as well as biochemical and structural alterations in the extracellular matrix macromolecules.     

Monitoring intermediate filament assembly by small-angle x-ray scattering reveals the molecular architecture of assembly intermediates

Intermediate filaments (IFs), along with microtubules, microfilaments, and associated cross-bridging proteins, constitute the cytoskeleton of metazoan cells. While crystallographic data on the dimer representing the elementary IF "building block" have recently become available, little structural detail is known about both the mature IF architecture and its assembly pathway. Here, we have applied solution small-angle x-ray scattering to investigate the in vitro assembly of a 53-kDa human IF protein vimentin at pH 8.4 by systematically varying the ionic strength conditions, and complemented these experiments by electron microscopy and analytical ultracentrifugation. While a vimentin solution in 5 mM Tris.HCl (pH 8.4) contains predominantly tetramers, addition of 20 mM NaCl induces further lateral assembly evidenced by the shift of the sedimentation coefficient and yields a distinct octameric intermediate. Four octamers eventually associate into unit-length filaments (ULFs) that anneal longitudinally. Based on the small-angle x-ray scattering experiments supplemented by crystallographic data and additional structural constraints, 3D molecular models of the vimentin tetramer, octamer, and ULF were constructed. Within each of the three oligomers, the adjacent dimers are aligned exclusively in an approximately half-staggered antiparallel A(11) mode with a distance of 3.2-3.4 nm between their axes. The ULF appears to be a dynamic and a relatively loosely packed structure with a roughly even mass distribution over its cross-section.     

Control of electrostatic interactions between F-actin and genetically modified lysozyme in aqueous media

The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin-lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin-lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin-lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity.