Multi-wavelength anomalous diffraction using medium-angle X-ray solution scattering (MADMAX)

Proteins are dynamic molecules whose function in virtually all biological processes requires conformational motion. Direct experimental probes of protein structure in solution are needed to characterize these motions. Anomalous scattering from proteins in solution has the potential to act as a precise molecular ruler to determine the positions of specific chemical groups or atoms within proteins under conditions in which structural changes can take place free from the constraints of crystal contacts. In solution, anomalous diffraction has two components: a set of cross-terms that depend on the relative location of the anomalous centers and the rest of the protein, and a set of pure anomalous terms that depend on the distances between the anomalous centers. The cross-terms are demonstrated here to be observable and to provide direct information about the distance between the anomalous center and the center of mass of the protein. The second set of terms appears immeasurably small in the context of current experimental capabilities. Here, we outline the theory underlying anomalous scattering from proteins in solution, predict the anomalous differences expected on the basis of atomic coordinate sets, and demonstrate the measurement of anomalous differences at the iron edge for solutions of myoglobin and hemoglobin.     

风花菜根异常次生结构的解剖学观察

利用光镜观察了风花菜（Rorippa islandica (Oed.)Borb.）的根的发育过程中的解剖结构的变化,发现风花菜根具有异常次生结构,第一年早期次生结构中,在导管周围形成额外形成层并由额外形成层向内外分裂形成大量的薄壁组织,从而使根快速增粗,在根的横切面上,导管分散在薄壁组织当中。第一年晚期的次生结构中,由木质部中薄壁组织细胞反分化形成额外形成层,许多分散的额外形成层片段连接形成形成层环,形成层环向内向外分裂形成三生木质部及三生韧皮部,从而构成三生维管组织环,三生维管组织环形成方向由外向内,可以形成多环。第二年风花菜的韧皮部以及皮层中的薄壁组织反分化形成额外形成层,并且由额外形成层片段连接形成额外形成层环,进而分裂形成三生维管组织环,而且在皮层中可以形成多层,形成的方向为由内向外。风花菜根不同发育时期的异常次生结构是与其生理活动相适应的。     

Cation-selective Pathway of OmpF Porin Revealed by Anomalous X-ray Diffraction

The OmpF porin from the Escherichia coli outer membrane folds into a trimer of β-barrels, each forming a wide aqueous pore allowing the passage of ions and small solutes. A long loop (L3) carrying multiple acidic residues folds into the β-barrel pore to form a narrow “constriction zone”. A strong and highly conserved charge asymmetry is observed at the constriction zone, with multiple basic residues attached to the wall of the β-barrel (Lys16, Arg42, Arg82 and Arg132) on one side, and multiple acidic residues of L3 (Asp107, Asp113, Glu117, Asp121, Asp126, Asp127) on the other side. Several computational studies have suggested that a strong transverse electric field could exist at the constriction zone as a result of such charge asymmetry, giving rise to separate permeation pathways for cations and anions. To examine this question, OmpF was expressed, purified and crystallized in the P6₃ space group and two different data sets were obtained at 2.6 Å and 3.0 Å resolution with K⁺ and Rb⁺, respectively. The Rb⁺-soaked crystals were collected at the rubidium anomalous wavelength of 0.8149 Å and cation positions were determined. A PEG molecule was observed in the pore region for both the K⁺ and Rb⁺-soaked crystals, where it interacts with loop L3. The results reveal the separate pathways of anions and cations across the constriction zone of the OmpF pore.     

Crystallographic structure analysis of lamprey hemoglobin from anomalous dispersion of synchrotron radiation

The molecular structure of lamprey hemoglobin was previously determined and refined by conventional crystallographic analysis. In this study, the structural analysis has been repeated in the course of developing the method of multiwavelength anomalous diffraction (MAD) for phase determination. New experimental and analytical procedures that were devised to perform this determination should have general applicability. These include an experimental design to optimize signal strength and reduce systematic errors, experimental evaluation of anomalous scattering factors, and a least-squares procedure for analyzing the MAD data. MAD phases for the structure at 3 A resolution are as accurate overall as the multiple isomorphous replacement (MIR) phases determined previously.     

Crystallization and preliminary X-ray characterization of the Methanothermus fervidus histones HMfA and HMfB

HMfA and HMfB are histone proteins from the thermophilic archaeon Methanothermus fervidus. They wrap DNA into nucleosome-like structures and appear to represent the basic core histone fold. HMfA was crystallized in space groups P4₂2₁2 and P2₁2₁2₁. HMfB crystallized in space group P2₁2₁2, while a selenomethionine-substituted variant, SeMet-HMfB, yielded crystals in C222₁. In all crystal forms HMfA, HMfB, or SeMet-HMfB may be present as homodimers.     

Defining the p53 DNA-binding domain/Bcl-x(L)-binding interface using NMR

We have determined the first de novo position of the secondary quinone Q_B in the Rhodobacter sphaeroides reaction center (RC) using phases derived by the single wavelength anomalous dispersion method from crystals with selenomethionine substitution. We found that in frozen RC crystals, Q_B occupies primarily the proximal binding site. In contrast, our room temperature structure showed that Q_B is largely in the distal position. Both data sets were collected in dark-adapted conditions. We estimate that the occupancy of the Q_B site is 80% with a proximal: distal ratio of 4:1 in frozen RC crystals. We could not separate the effect of freezing from the effect of the cryoprotectants ethylene glycol or glycerol. These results could have far-reaching implications in structure/function studies of electron transfer in the acceptor quinone complex because the above are the most commonly used cryoprotectants in spectroscopic experiments.     

Structural analysis of a ternary complex of allantoate amidohydrolase from Escherichia coli reveals its mechanics

Purine metabolism plays a major role in regulating the availability of purine nucleotides destined for nucleic acid synthesis. Allantoate amidohydrolase catalyzes the conversion of allantoate to (S)-ureidoglycolate, one of the crucial alternate steps in purine metabolism. The crystal structure of a ternary complex of allantoate amidohydrolase with its substrate allantoate and an allosteric effector, a sulfate ion, from Escherichia coli was determined to understand better the catalytic mechanism and substrate specificity. The 2.25 A resolution X-ray structure reveals an alpha/beta scaffold akin to zinc exopeptidases of the peptidase M20 family and lacks the (beta/alpha)(8)-barrel fold characteristic of the amidohydrolases. Arrangement of the substrate and the two co-catalytic zinc ions at the active site governs catalytic specificity for hydrolysis of N-carbamyl versus the peptide bond in exopeptidases. In its crystalline form, allantoate amidohydrolase adopts a relatively open conformation. However, structural analysis reveals the possibility of a significant movement of domains via rotation about two hinge regions upon allosteric effector and substrate binding resulting in a closed catalytically competent conformation by bringing the substrate allantoate closer to co-catalytic zinc ions. Two cis-prolyl peptide bonds found on either side of the dimerization domain in close proximity to the substrate and ligand-binding sites may be involved in protein folding and in preserving the integrity of the catalytic site.     

Crystallinity and structure of starch using wide angle X-ray scattering

Wide angle X-ray diffraction was used to evaluate the crystalline fraction of a variety of starches, using preliminary smoothing then an iterative smoothing algorithm to estimate amorphous background scattering. This methodology was then used to determine initial crystallinity and monitor gelation and retrogradation of high amylose thermoplastic starch used to produce film. Retrogradation was monitored over a 5-day period. It was found that the starch film retrograded rapidly over the first 12 h with the film displaying both B-type crystallinity and long range amorphous ordering that were separately quantitatively calculated. Changes in starch films, including complete or partial gelatinization, retrogradation and crystallinity, were all determined through wide angle X-ray diffraction.     

Comparison of the structure of ribosomal 5S RNA from E. coli and from rat liver using X-ray scattering and dynamic light scattering

The structures of eukaryotic ribosomal 5S RNA from rat liver and of prokaryotic 5S RNA from E. coli (A-conformer) have been investigated by scattering methods. For both molecules, a molar mass of 44,500±4,000 was determined from small angle X-ray scattering as well as from dynamic light scattering. The shape parameters of the two rRNAs, volume V _c, surface O _c, radius of gyration R _s, maximum dimension of the molecule L, thickness D, and cross section radius of gyration R _sq, agree within the experimental error limits. The mean values are V _c=57±3 nm³, O _c=165±10 nm², R _s=3.37±0.05 nm, L=10.8±0.7 nm, D=1.57±0.07 nm, R _sa=0.92±0.01 nm.Identical structures for the E. coli 5S rRNA and the rat liver 5S rRNA at a resolution of 1 nm can be deduced from this agreement and from the comparison of experimental X-ray scattering curves and of experimental electron distance distribution function. The flat shape model derived for prokaryotic and eukaryotic 5S rRNA shows a compact region and two protruding arms. Double helical stems are eleven-fold helices with a mean base pair distance of 0.28 nm. Combining the shape information obtained from X-ray scattering with the information about the frictional behaviour of the molecules, deduced from the diffusion coefficients D _20,w ⁰ =(5.9±0.2)·10^-7 cm²s^-1 and (6.2±0.2)·10^-7 cm²s^-1 for rat liver 5S rRNA and E. coli 5S rRNA, respectively, a solvation shell of about 0.3 nm thickness around both molecules is determined. This structural similarity and the consensus secondary structure pattern derived from comparative sequence analyses suggest that all 5S rRNAs may indeed have conserved essentially the same type of folding of their polynucleotide strands during evolution, despite having very different sequences.     

Conformational stability of peanut agglutinin using small angle X-ray scattering

In this work, quaternary conformational studies of peanut agglutinin (PNA) have been carried out using small-angle X-ray scattering (SAXS). PNA was submitted to three different conditions: pH variation (2.5, 4.0, 7.4 and 9.0), guanidine hydrochloride presence (0.5-2M) at each pH value, and temperature ranging from 25 to 60°C. All experiments were performed in the absence and presence of T-antigen to evaluate its influence on the lectin stability. At room temperature and pH 4.0, 7.4 and 9.0, the SAXS curves are consistent with the PNA scattering in its crystallographic native homotetrameric structure, with monomers in a jelly roll fold, associated by non-covalent bonds resulting in an open structure. At pH 2.5, the results indicate that PNA tends to dissociate into smaller sub-units, as dimers and monomers, followed by a self-assembling into larger aggregates. Furthermore, the conformational stability under thermal denaturation follows the pH sequence 7.4>9.0>4.0>2.5. Such results are consistent with the conformational behavior found upon GndHCl influence. The presence of T-antigen does not affect the protein quaternary structure in all studied systems within the SAXS resolution.     

The development of concepts of chiral discrimination

Pasteur's conjecture (1860) that biomolecular homochirality arose from a chiral natural force as yet inaccessible in the laboratory was supplanted by Fischer's (1894) "key and lock" hypothesis of stereoselection in enantiomer to diastereomer conversions, whether in the laboratory or in living organisms. Elaborations of the "key and lock" hypothesis by Haldane (1930) and Pauling (1948) have been illustrated and supported with modification by X-ray diffraction crystal structures of enzyme-substrate complexes over the past quarter century. Two types of mechanism for the product diastereoselectivity in the reactions of an enantiomer with an achiral reagent, early proposed, have recent support: one proposes a quasidiastereomeric structure for the enantiomer attacked in the ground state, the other for the corresponding transition state of the reaction. Approaches to the differential biological activity of two enantiomers postulate either the complete binding of each isomer to a chiral receptor site, resulting in diastereomeric complexes with inequivalent bioactivities, or the differential binding of the two isomers to a set of three sites, with which only one isomer is sterically congruent. Biochemical homochirality, based on the chiral stereoselectivity of both biosynthetic and metabolic reactions, derives from the evolutionary pressure for a progressive enhancement of the kinetic efficiency and economy of those reactions. Recently Pasteur has been vindicated in part, and the problem of the original prebiotic enantiomeric excess left outstanding by Fischer has been solved. The unification of the electromagnetic with the weak interaction provided a universal chiral natural force, the electroweak interaction, which favours the chiral series selected during the course of biochemical evolution, both the D-sugars and the L-amino acids.     

Determination of absolute configuration using vibrational circular dichroism spectroscopy: the chiral sulfoxide 1-thiochromanone S-oxide

We reexamined the absolute configuration (AC) of the chiral sulfoxide 1-thiochromanone S-oxide (1) using vibrational circular dichroism (VCD) spectroscopy. The VCD spectrum of 1 was analyzed using density functional theory (DFT). DFT predicts two stable conformations of 1, separated by <1 kcal/mole. Their VCD spectra were calculated using the DFT/GIAO methodology. The VCD spectrum predicted for the equilibrium mixture of the two conformations of (S)-1 is in excellent agreement with the experimental spectrum of (+)-1. The AC of 1 is therefore definitively R(-)/S(+).     

Combinatorial evaluation of the chiral discrimination of permethylated carbohydrates using fast-atom bombardment mass spectrometry

The chiral discrimination abilities of several variously permethylated carbohydrates toward various amino acid 2-propyl esters were combinatorially evaluated from the relative peak intensity of the 1:1 diastereomeric complex ions with the deuterium-labeled L-amino acid 2-propyl ester protonated ion and with the unlabeled D-amino acid 2-propyl ester protonated ions in FAB mass spectrometry. The chiral discrimination abilities evaluated using FAB mass spectrometry approximately corresponded to the ratio of the association constants (K(R)/K(S)) toward each enantiomer in the solution. Therefore, this evaluation method is very useful for the screening of the chiral discrimination abilities of carbohydrates and their derivatives.     

Palladium(II) complexes of the chiral pinBPI ligand

The chiral bis(pyridylimino)isoindoline derivative 5 (pinBPI) was prepared from the chiral pool reagent α-pinene 1 in three steps and an overall yield of 12%. Upon treatment with palladium(II) acetate and palladium(II) chloride, pinBPI 5 forms the nonracemic chiral complexes [(pinBPI)Pd(OAc)] 6 and [(pinBPI)PdCl] 7, respectively, as the only isolated species in good yield. The synthesis of a related thiazole-based ligand failed for the incompatibility of the four-membered carbocycle of the pinene subunit with the conditions of the thiazole-forming reaction. Both pinBPI chelates crystallize in chiral space groups but with different molecular conformations and intermolecular interactions. A triclinic system with space group P1 is found for the acetato derivative 6 which organizes pseudosymmetrically as a chloroform solvate with one helical and one (almost) planar molecule in the asymmetric unit. The chloro derivate 7 on the other hand crystallizes in the monoclinic space group P2₁ with Z = 8 and four independent molecules in the unit cell. Here, all molecules are in a pseudoplanar configuration with convex ligand conformations, but differ significantly in bond lengths and angles. The structures unravel two different possible scenarios for intermolecular association of such chiral BAI ligands.     

Prediction of protein conformational mobility with validation using small-angle X-ray scattering

A “coarse-grained” model of protein conformational mobility is presented. The conformational paths in five proteins, predicted using the model, are compared with those obtained by the nearest-neighbor method basing on the small-angle X-ray scattering data. The sequences of conformations evaluated with the help of these two approaches have been shown for all proteins under consideration to coincide well; yet there are exceptions, their causes having to be considered for each protein separately.