Improved purification of native meningococcal porin PorB and studies on its structure/function

The outer membrane protein PorB of Neisseria meningitidis is a pore-forming protein which has various effects on eukaryotic cells. It has been shown to (1) up-regulate the surface expression of the co-stimulatory molecule CD86 and of MHC class II (which are TLR2/MyD88 dependent and related to the porin's immune-potentiating ability), (2) be involved in prevention of apoptosis by modulating the mitochondrial membrane potential, and (3) form pores in eukaryotic cells. As an outer membrane protein, its native trimeric form isolation is complicated by its insoluble nature, requiring the presence of detergent throughout the whole procedure, and by its tight association with other outer membrane components, such as neisserial LOS or lipoproteins. In this study, an improved chromatographic purification method to obtain an homogeneous product free of endotoxin and lipoprotein is described, without loss of any of the above-mentioned properties of the porin. Furthermore, we have investigated the requirement of the native trimeric structure for the porin's activity. Inactivation of functional PorB trimers into non-functional monomers was achieved by incubation on ice. Thus, routine long- and medium-term storage at low temperature may be a cause of porin inactivation.     

Chiral separation and CD characterisation of enantiomeric cyclotriphosphazene derivatives

The gem-disubstituted cyclotriphosphazene 1 reacted with piperazine (pip) to give the piperazine-bridged derivative 2, which is expected to exist in meso and racemic forms because the two PCl (pip) groups are stereogenic. The proton-decoupled (31)P NMR spectrum of 2 gave rise to two similar sets of ABX signals in a 1:1 ratio, consistent with formation of diastereoisomers. The meso and racemic forms of compound 2 were separated by column chromatography on silica gel and characterised by elemental analysis, mass spectrometry, (31)P NMR spectroscopy, and X-ray crystallography. Using HPLC with a chiral stationary phase, the racemic form of compound 2 was further separated into enantiomers, which were characterised by circular dichroism (CD) spectroscopy. This is the first report of the separation of enantiomers in the field of cyclophosphazene chemistry and hence the first CD spectra of derivatives in which the cyclophosphazene ring is at the chiral centre.     

Estimating the Accuracy of Protein Structures using Residual Dipolar Couplings

It has been commonly recognized that residual dipolar coupling data provide a measure of quality for protein structures. To quantify this observation, a database of 100 single-domain proteins has been compiled where each protein was represented by two independently solved structures. Backbone ¹H–¹⁵N dipolar couplings were simulated for the target structures and then fitted to the model structures. The fits were characterized by an R-factor which was corrected for the effects of non-uniform distribution of dipolar vectors on a unit sphere. The analyses show that favorable values virtually guarantee high accuracy of the model structure (where accuracy is defined as the backbone coordinate rms deviation). On the other hand, unfavorable values do not necessarily suggest low accuracy. Based on the simulated data, a simple empirical formula is proposed to estimate the accuracy of protein structures. The method is illustrated with a number of examples, including PDZ2 domain of human phosphatase hPTP1E. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Atomically detailed potentials to recognize native and approximate protein structures

Atomically detailed potentials for recognition of protein folds are presented. The potentials consist of pair interactions between atoms. One or three distance steps are used to describe the range of interactions between a pair. Training is carried out with the mathematical programming approach on the decoy sets of Baker, Levitt, and some of our own design. Recognition is required not only for decoy-native structural pairs but also for pairs of decoy and homologous structures. Performance is tested on the targets of CASP5 using templates from the Protein Data Bank, on two test ab initio decoy sets from Skolnick's laboratory, and on decoy sets from Moult's laboratory. We conclude that the newly derived potentials have significant recognition capacity, comparable to the best models derived from other techniques. The new potentials require a significantly smaller number of parameters. The enhanced recognition capacity extends primarily to the identification of structures generated by ab initio simulation and less to the recognition of approximate shapes created by homology.     

Comparison of X‐ray and NMR structures: Is there a systematic difference in residue contacts between X‐ray‐ and NMR‐resolved protein structures?

We have compared structures of 78 proteins determined by both NMR and X-ray methods. It is shown that X-ray and NMR structures of the same protein have more differences than various X-ray structures obtained for the protein, and even more than various NMR structures of the protein. X-ray and NMR structures of 18 of these 78 proteins have obvious large-scale structural differences that seem to reflect a difference of crystal and solution structures. The other 60 pairs of structures have only small-scale differences comparable with differences between various X-ray or various NMR structures of a protein; we have analyzed these structures more attentively. One of the main differences between NMR and X-ray structures concerns the number of contacts per residue: (1) NMR structures presented in PDB have more contacts than X-ray structures at distances below 3.0 A and 4.5-6.5 A, and fewer contacts at distances of 3.0-4.5 A and 6.5-8.0 A; (2) this difference in the number of contacts is greater for internal residues than for external ones, and it is larger for beta-containing proteins than for all-alpha proteins. Another significant difference is that the main-chain hydrogen bonds identified in X-ray and NMR structures often differ. Their correlation is 69% only. However, analogous difference is found for refined and rerefined NMR structures, allowing us to suggest that the observed difference in interresidue contacts of X-ray and NMR structures of the same proteins is due mainly to a difference in mathematical treatment of experimental results.     

Can morphogenesis be understood in terms of physical rules?

Because the morphogenesis of biological systems is not fully understood, researches from various points of view are necessary. The present author has recently made computer simulations with his colleagues to construct branching systems of human organs, such as the lung airway and the liver blood vessels. In the simulations certain rules are assumed to govern bifurcating processes of the systems. These rules are expressed in terms of physical and geometrical concepts, such as minimum energy consumption and uniform filling of branches in the space of organs. Results of computer simulation are quite similar to real structures. However, actual mechanisms of morphogenesis, i.e. effects of genes or proteins, are not considered in these studies. In this article, the present work is discussed in relation to the concept of biological pattern formation by Meinhardt and a recent study by Miura and Shiota on lung growth.     

Biodiversity analysis of microbial community in the chem-bioflocculation treatment process

Total DNA was directly extracted from environmental samples and amplified with polymerase chain reaction (PCR) technique. The PCR products were fingerprinted via denaturing gradient gel electrophoresis (DGGE). Significant differences were observed in the microbial community structures between traditional treatment process and chem-bioflocculation process. The microbial community structure shift at different sampling locations in chem-bioflocculation process and on two typical operational conditions was studied. 16S rDNA V3 regions of some dominant species were sequenced and the species were identified. The microbial communities were stable in both the chem-bioflocculation process and the activated sludge process under various experimental conditions presented in this work. The attached growth treatment process was less stable when operational conditions changed.     

Fluorous interactions in complexes of lead(II) hexafluoroacetylacetonate

Structure determinations for 2,2′-bipyridine and 1,10-phenanthroline adducts of lead(II) hexafluoroacetylacetonate, [Pb(bipy)₂(hfacac)₂] (1), [Pb(bipy)(hfacac)₂] (2), and [Pb(phen)(hfacac)₂] (3), show that the balance of intermolecular forces within the lattices is seemingly sensitive to the adduct stoichiometry but not to the nature of the heteroaromatic base. In 3, a structure, in which there is an apparent preference for CF/aromatic interactions over separate CF/CF and aromatic/aromatic interactions, is essentially identical at both 120 and 293 K.     

Synthesis and characterisation of palladium(II) iodo complexes containing water soluble phosphine ligands. Crystal structures of [PdI2(PTA-H)2][PtI3(PTA)]2 · 2H2O and trans-[PdI2(PTA)2]

The aqueous solution behaviour of the equilibrium related cis-[PdCl₂(PTA)₂] and [PdCl(PTA)₃]Cl complexes has been investigated in the presence of acid and iodide ions. Several of the resulting species were identified and a reaction scheme accounting for identified complexes is proposed. The crystal structures of trans-[PdI₂(PTA-H)₂][PdI₃(PTA)]₂ · 2H₂O (1) (PTA-H⁺ = protonated form of PTA) and trans-[PdI₂(PTA)₂] (2) are reported. The geometry around the Pd(II) metal centre in 1 (for both the cation and anion) and 2 is distorted square planar. The PTA ligands occupy a trans orientation in the cation of 1 and in complex 2. Compound 1 represents a rare example of a Pd(II) system wherein the cation:anion pair, in a 1:2 ratio, are both coordination complexes. It is the first d⁸ Ni-triad square planar complex containing only one PTA ligand and only the second platinum group metal complex. For the cation in 1, the bond distances and angles are Pd(1)-P(1) = 2.2864(16) Å, Pd(1)-I(1) = 2.6216(7) Å, P(1)-Pd(1)-P(1)′ = 180.00(7)° and P(1)-Pd(1)-I(1) = 87.62(4)°, while in the anion the bond distances are Pd(2)-P(2) = 2.2377(15) Å, Pd(2)-I(4) = 2.5961(13) Å, Pd(2)-I(2) = 2.6328(13) Å, Pd(2)-I(3) = 2.6513(8) Å, while the angles are P(2)-Pd(2)-I(4) = 90.00(5)°, P(2)-Pd(2)-I(2) = 89.69(5)°, I(4)-Pd(2)-I(2) = 179.57(2)°, P(2)-Pd(2)-I(3) = 175.19(4)°, I(4)-Pd(2)-I(3) = 90.29(4)° and I(2)-Pd(2)-I(3) = 90.05(4)°. Bond distances and angles of the coordination polyhedron in 2 are Pd-P = 2.327(3) Å, Pd-I = 2.5916(10) Å, P-Pd-I = 89.13(7)° and P-Pd-P = 180.00(13)°. The average effective- and Tolman cone angles for the two ligands, calculated from the crystallographic data, are 115° and 117° for PTA and PTA-H, respectively.     

Hydrogen bonding in diruthenium(II,III) tetraacetate complexes with biologically relevant axial ligands

Three diadduct complexes of the mixed-valent form of diruthenium tetraacetate, [Ru₂(μ-O₂CCH₃)₄L₂](PF₆), where L are the biologically relevant ligands imidazole, 1, 7-azaindole, 2, and caffeine, 3, were synthesized and characterized by elemental analysis, IR and UV-Vis spectroscopy and X-ray crystallography. In order to further elucidate the potential interactions of these dimers with DNA, the nature of the ligand coordination and the secondary inter- and intramolecular hydrogen-bonding interactions in all three complexes were assessed. Complex 1 · CH₂Cl₂ shows, exclusively, intermolecular interactions with the counterion whereas complexes 2 · ClCH₂CH₂Cl and 3 · OC(CH₃)₂ · H₂O, in addition to extensive intermolecular interactions, show intramolecular hydrogen bonding from the axial ligand to the bridging acetate oxygens, locking the ligand mean planes in place between the bridging acetate mean planes. In addition, all three complexes display π-π stacking of axial ligand rings on adjacent diadduct units.