A. Van Dorsselaer

Characterisation of the heptameric pore‐forming complex of the Aeromonas toxin aerolysin using MALDI‐TOF mass spectrometry

Aerolysin, a virulence factor secreted by Aeromonas hydrophila, is representative of a group of β‐sheet toxins that must form stable homooligomers in order to be able to insert into biological membranes and generate channels. Electron microscopy and image analysis of two‐dimensional membrane crystals had previously revealed a structure with 7‐fold symmetry et al. (1992) EMBO J. 11, 2457–2463]. However, this unusual et al. (1992) EMBO J. 11, 2457–2463]. However, this unusual molecularity of the channel remained to be confirmed by an independent method since low‐resolution electron crystallography had led to artefactual data for other pore‐forming toxins. In this study, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) was used to measure the mass of the aerolysin oligomer preparation. A mass of 333 850 Da was measured, fitting very well with a heptameric complex (expected mass: 332 300 Da). These results confirm the earlier evidence that the aerolysin oligomer is a heptamer and also show that MALDI‐TOF mass spectrometry could be a valuable tool to study non‐covalent association of proteins.

PURIFICATION AND CHARACTERIZATION OF ANDROGENIC HORMONE FROM THE TERRESTRIAL ISOPOD ARMADILLIDIUM VULGARE LATR. (CRUSTACEA, ONISCIDEA)

Androgenic hormone (AH) was purified from hypertrophied androgenic glands of intersexed Armadillidium vulgare (genetic males feminized by symbiotic endocellular bacteria). Two isohormones labeled AH1 and AH2 with similar molecular weights in the range 17,000-18,000 were isolated. Amino acid analysis showed the absence of cysteine in these two forms. A polyclonal antiserum was raised which recognized AH1 and AH2. The physiological significance of this polymorphism is still not known.

High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase.

Neutron diffraction data have been collected to 2.2 Angstrom resolution from a small (0.15 mm(3)) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm(3) are reported. Neutron data were recorded to 2 Angstrom resolution, with subsequent data analysis using data to 2.2 Angstrom. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the subunit stoichiometry study of high mass non-covalent complexes

This study explores the potential of MALDI-TOF MS for the mass measurement of large non-covalent protein complexes. The following non-covalent complexes have been investigated: aerolysin from Aeromonas hydrophila (335 kDa) and α-haemolysin from Staphylococcus aureus (233 kDa) which are both cytolytic toxins, three enzymes known to be homotetramers in solution: bovine liver catalase (235 kDa), rabbit muscle pyruvate kinase (232 kDa), yeast alcohol dehydrogenase (147 kDa) and finally a lectin, concanavalin A (102 kDa). Three different matrix preparations were systematically tested under various conditions: ferulic acid dissolved in THF, 2,6-dihydroxyacetophenone in 20 mM aqueous ammonium citrate and a two-step sample preparation with sinapinic acid. It was possible to find a suitable combination of matrix and preparation type which allowed the molecularity of all complexes tested to be deduced from the MALDI mass spectrum. Trimeric and tetrameric intermediates accumulating during the formation of the active heptameric aerolysin complex were also identified, this allowing a formation mechanism to be proposed. The observation of large specific non-covalent complexes has been found to be dependent on the choice of matrix, the type of sample preparation used, the solvent evaporation speed, the pH of the resulting matrix-sample mixture and the number of shots acquired on a given area. From this set of experiments, some useful guidelines for the observation of large complexes by MALDI could therefore be deduced. Fast evaporation of the solvent is particularly necessary in the case of pH sensitive complexes. An ESMS study on the same non-covalent complexes indicated that, rather surprisingly, reliable results could be obtained by MALDI-TOF MS on several very large complexes (above 200 kDa) for which ESMS yielded no clear spectra.

Production of crystals of human aldose reductase with very high resolution diffraction

As the action of human aldose reductase (hAR) is thought to be linked to the pathogenesis of diabetic complications, much effort has been directed towards the analysis of the catalytic mechanism and the development of specific inhibitors. Here, the crystallization of recombinant hAR with its cofactor NADP+ at 277 K in the presence of the precipitating agent PEG 6000 is reported. The crystals diffract to high resolution (1.1 A) and belong to the P21 space group with unit-cell parameters a = 49.97, b = 67.14, c = 48. 02 A, beta = 92.2 degrees with one molecule per asymmetric unit. Seleno-substituted hAR crystals were also produced and diffract to 1. 7 A on a conventional X-ray source.

Aah VI, a novel, N-glycosylated anti-insect toxin from Androctonus australis hector scorpion venom: isolation, characterisation, and glycan structure determination

Aah VI was isolated from the venom of the North African scorpion, Androctonus australis hector. It is the first glycosylated neurotoxin from scorpion venom to be described. It was not toxic to mice, when injected intracerebroventricularly at a dose of 1.2 μg per animal. However, it had typical activity in Blatella germanica cockroaches resulting in gradual paralysis and very low toxicity (LD50=8.5 μg/g of animal). It consists of 66 amino acid residues and is heterogeneously N‐glycosylated at a single site, on asparagine 9, of the Asn‐Gly‐Thr sequence. The potential N‐glycosylation site was deduced from automatic Edman degradation and amino acid analysis, and glycan heterogeneity was evidenced by ESMS. Determination of the N‐glycan structures (dHex, Hex and HexNAc) was assessed by nanoESMS/MS with picomolar amounts of sample. Current knowledge of N‐glycan structure and composition suggests that the glycan structures are derived from a common core.

Crystallization of Previously Desalted Lysozyme in the Presence of Sulfate Ions

Lysozyme, which is known to crystallize readily in the presence of many salts, has never been crystallized by salting out with ammonium sulfate. In the present study, lysozyme was first completely desalted by treatment with strong cation- (H(+) form) and anion- (OH(-) form) exchange resins. This leads to a protein solution with only H(+) and OH(-) as counterions, corresponding to its isoionic point. Addition of 2.5-3 molar equivalents of H(2)SO(4) to isoionic lysozyme decreases the pH value to 9-8 and allows crystallization to take place. The space group was found to be P4(3)2(1)2, similar to the classical lysozyme crystals grown in the presence of NaCl at pH 4.5, with unit-cell dimensions a = b = 78.9, c = 38.5 A. Tentative explanation of the sulfate/lysozyme interaction was addressed by mass spectrometry, and shows non-covalent binding of the ions on the protein.

Endogenous morphine-like compound immunoreactivity increases in parkinsonism

Morphine is endogenously synthesized in the central nervous system and endogenous dopamine is thought to be necessary for endogenous morphine formation. As Parkinsons disease results from the loss of dopamine and is associated with central pain, we considered how endogenous morphine is regulated in the untreated and l-DOPA-treated parkinsonian brain. However, as the cellular origin and overall distribution of endogenous morphine remains obscure in the pathological adult brain, we first characterized the distribution of endogenous morphine-like compound immunoreactive cells in the rat striatum. We then studied changes in the endogenous morphine-like compound immunoreactivity of medium spiny neurons in normal, Parkinsons disease-like and l-DOPA-treated Parkinsons disease-like conditions in experimental (rat and monkey) and human Parkinsons disease. Our results reveal an unexpected dramatic upregulation of neuronal endogenous morphine-like compound immunoreactivity and levels in experimental and human Parkinsons disease, only partially normalized by l-DOPA treatment. Our data suggest that endogenous morphine formation is more complex than originally proposed and that the parkinsonian brain experiences a dramatic upregulation of endogenous morphine immunoreactivity. The functional consequences of such endogenous morphine upregulation are as yet unknown, but based upon the current knowledge of morphine signalling, we hypothesize that it is involved in fatigue, depression and pain symptoms experienced by patients with Parkinsons disease.

Characterization of crystal content by ESI–MS and MALDI–MS

A general approach based on mass spectrometry is described for the rapid identification of the content of macromolecular crystals. The experimental procedure was established using lysozyme crystals and then successfully applied to various systems containing specifically bound molecules not easily detectable by other classical techniques. This procedure can be carried out on crystals containing macromolecules of a different nature, such as proteins, nucleic acids and small organic molecules and their non-covalent complexes, grown under various crystallization conditions including PEGs and salts. It can be applied very early on in the crystallization process - as soon as the crystals can be handled. It allows the biologist to control precisely the sequence integrity and homogeneity of the crystallized proteins (in particular at the C-terminus) as well as to verify whether the protein has crystallized with all its expected partners or ligands (nucleic acid molecules, cofactor or small organic molecules).

Isolation and amino acid sequence of a novel 6.8-kDa mitochondrial proteolipid from beef heart : use of FAB-MS for molecular mass determination

We have isolated a 6.8 kDa proteolipid from an acidic chloroform/methanol extract of bovine cardiac muscle. The molecular mass of the polypeptide was measured by fast atom bombardment‐mass spectrometry (FAB‐MS) (m/z 6834.1). Its amino acid sequence was partly determined by direct sequencing and completed by characterization of cyanogen bromide and tryptic fragments (sequencing, FAB‐MS and amino acid analysis). The polypeptide consists of 60 amino acid residues. Polyclonal antibodies raised in rabbit allowed its localization by electroimmunoblotting in mitochondria.