Globin's structure and function in vesicomyid bivalves from the Gulf of Guinea cold seeps as an adaptation to life in reduced sediments.

Vesicomyid bivalves form dense clam beds in both deep-sea cold seeps and hydrothermal vents. The species diversity within this family raises questions about niche separation and specific adaptations. To compare their abilities to withstand hypoxia, we have studied the structure and function of erythrocyte hemoglobin (Hb) and foot myoglobin (Mb) from two vesicomyid species, Christineconcha regab and Laubiericoncha chuni, collected from the Regab pockmark in the Gulf of Guinea at a depth of 3,000 m. Laubiericoncha chuni possesses three monomeric globins, G1 (15,361 Da), G2 (15,668 Da), and G3 (15,682 Da) in circulating erythrocytes (Hb), and also three globins, G1, G3, and G4 (14,786 Da) in foot muscle (Mb). Therefore, globins G2 and G4 appear to be specific for erythrocytes and muscle, respectively, but globins G1 and G3 are common. In contrast, C. regab lacks erythrocyte Hb completely and possesses only globin monomers G1' (14,941 Da), G2' (15,169 Da), and G3' (15,683 Da) in foot muscle. Thus, these two vesicomyid species, C. regab and L. chuni, show a remarkable diversity in globin expression when examined by electrospray ionization mass spectrometry. Oxygen-binding affinities reveal extremely high oxygen affinities (P50 < 1 Torr, from 5° to 15°C at pH 7.5), in particular L. chuni globins, which might be an advantage allowing L. chuni to dig deeply for sulfides and remain buried for long periods in reduced sediments.

A unique PPARgamma ligand with potent insulin-sensitizing yet weak adipogenic activity.

FMOC-L-Leucine (F-L-Leu) is a chemically distinct PPARgamma ligand. Two molecules of F-L-Leu bind to the ligand binding domain of a single PPARgamma molecule, making its mode of receptor interaction distinct from that of other nuclear receptor ligands. F-L-Leu induces a particular allosteric configuration of PPARgamma, resulting in differential cofactor recruitment and translating in distinct pharmacological properties. F-L-Leu activates PPARgamma with a lower potency, but a similar maximal efficacy, than rosiglitazone. The particular PPARgamma configuration induced by F-L-Leu leads to a modified pattern of target gene activation. F-L-Leu improves insulin sensitivity in normal, diet-induced glucose-intolerant, and in diabetic db/db mice, yet it has a lower adipogenic activity. These biological effects suggest that F-L-Leu is a selective PPARgamma modulator that activates some (insulin sensitization), but not all (adipogenesis), PPARgamma-signaling pathways.

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).

Determination by electrospray mass spectrometry of the outersphere association constants of DNA/platinum complexes using 20-mer oligonucleotides and ([Pt(NH(3))(4)](2+), 2Cl(-)) or ([Pt(py)(4)](2+), 2Cl(-)).

The cytotoxic effects of cisplatin, cis-diamminedichloroplatinum(II), are generally ascribed to the formation of DNA adducts. Several in vitro as well as in vivo studies showed that cisplatin binds preferentially to guanines belonging to (G)(n) sequences (n > or = 2). After mono- or diaquation of cisplatin, giving the cationic complexes cis-[PtCl(NH(3))(2)(H(2)O)](+) and cis-[Pt(NH(3))(2)(H(2)O)(2)](2+), DNA platination occurs in two steps: the cationic complex gives an outersphere association with DNA and the actual coordination then occurs by substitution of one aqua ligand by guanine-N7. For a better understanding of the (G)(n) selectivity of cisplatin giving the biologically active adducts, also necessary for the conception of new platinum drugs, the respective contribution of the outersphere association and actual guanine platination must be investigated. To check the role of outersphere association in the overall platination process, we used electrospray mass spectrometry (ESMS) to detect and quantify outersphere association between 20-mer oligonucleotides and platinum complexes. The 20-mer oligonucleotides were single- or double-stranded, with the same number of guanines either isolated or adjacent to each other. To deal only with outersphere association and check the influence of platinum ligands, the [Pt(NH(3))(4)](2+) and [Pt(py)(4)](2+) complexes were used. We characterized by ESMS all the different outersphere association species formed during titration of each oligonucleotide with the various platinum complexes and evaluated their affinity constants. The ESMS results demonstrate that the outersphere association depends on electrostatic interactions and on the ability of the platinum ligands to participate to hydrogen bonding, particularly within the duplex form.

Heterodimeric complex of RAR and RXR nuclear receptor ligand-binding domains: purification, crystallization, and preliminary X-ray diffraction analysis.

Both the human retinoic acid receptor alpha (hRARalpha) and a constitutively active mutant (F318A) of the mouse retinoid X receptor alpha (mRXR alpha F318A) ligand-binding domains were separately overexpressed in Escherichia coli, copurified as a heterodimer in a two-step procedure, and cocrystallized with an RAR alpha-specific antagonist by using polyethylene glycol 10,000 as precipitant. The crystals grew in the hexagonal space group P6(1)22 displaying the unit cell parameters a = b = 116.6 A and c = 207.8 A. They diffracted X-ray to a limit of 2.2-A resolution. The asymmetric unit comprises one heterodimer and the crystal contains 60% solvent. The structure was determined by molecular replacement and is currently being refined.

Binding of aldose reductase inhibitors: correlation of crystallographic and mass spectrometric studies.

Aldose reductase is a NADP(H)-dependent enzyme, believed to be strongly implicated in the development of degenerative complications of Diabetes Mellitus. The search for specific inhibitors of this enzyme has thus become a major pharmaceutic challenge. In this study, we applied both X-ray crystallography and mass spectrometry to characterize the interactions between aldose reductase and four representative inhibitors: AminoSNM, Imirestat, LCB3071, and IDD384. If crystallography remains obviously the only way to get an extensive description of the contacts between an inhibitor and the enzymatic site, the duration of the crystallographic analysis makes this technique incompatible with high throughput screenings of inhibitors. On the other hand, dissociation experiments monitored by mass spectrometry permitted us to evaluate rapidly the relative gas-phase stabilities of the aldose reductase-inhibitor noncovalent complexes. In our experiments, dissociation in the gas-phase was provoked by increasing the accelerating voltage of the ions (Vc) in the source-analyzer interface region: the Vc value needed to dissociate 50% of the noncovalent complex initially present (Vc50) was taken as a gas-phase stability parameter of the enzyme-inhibitor complex. Interestingly, the Vc50 were found to correlate with the energy of the electrostatic and H-bond interactions involved in the contact aldose reductase/inhibitor (Eel-H), computed from the crystallographic model. This finding may be specially interesting in a context of drug development. Actually, during a drug design optimization phase, the binding of the drug to the target enzyme is often optimized by modifying its interatomic electrostatic and H-bond contacts; because they usually depend on a single atom change on the drug, and are easier to introduce than the hydrophobic interactions. Therefore, the Vc50 may help to monitor the chemical modifications introduced in new inhibitors. X-ray crystallography is clearly needed to get the details of the contacts and to rationalize the design. Nevertheless, once the cycle of chemical modification is engaged, mass spectrometry can be used to select a priori the drug candidates which are worthy of further crystallographic investigation. We thus propose to use the two techniques in a complementary way, to improve the screening of large collections of inhibitors.

Study of a noncovalent trp repressor: DNA operator complex by electrospray ionization time-of-flight mass spectrometry.

Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) has been used to study noncovalent interactions between the trp apo-repressor (TrpR), its co-repressor tryptophan and its specific operator DNA. In 5 mM ammonium acetate, TrpR was detected as a partially unfolded monomer. In the presence of a 21-base-pair DNA possessing the two symmetrically arranged CTAG consensus sequences required for specific TrpR binding, a homodimer-dsDNA complex with a 1:1 stoichiometry was observed. Co-repressor was not needed for the complex to form under our experimental conditions. Collision induced dissociation (CID-MS) revealed that this complex was very stable in the gas phase since dissociation was achieved only at energies that also broke covalent bonds. We saw no evidence for the presence of the six water molecules that mediate the interaction between the protein and the DNA in the crystal structure. To check the binding specificity of the TrpR for its target DNA, a competitive experiment was undertaken: the protein was mixed with an equimolar amount of three different DNAs in which the two CTAG sequences were separated by 2, 4, and 6 bp, respectively. Only the DNA with the correct consensus spacing of 4 bp was able to form stable interactions with TrpR. This experiment demonstrates the potential of ESI-MS to test the sequence-specificity of protein-DNA complexes. The interactions between the TrpR-DNA complex and 5-methyl-, L- and D-tryptophan were also investigated. Two molecules of 5-methyl- or L-tryptophan were bound with high affinity to the TrpR-DNA complex. On the other hand, D-tryptophan appeared to bind to the complex with poor specificity and poor affinity.

Mechanisms of inhibition of the thioredoxin growth factor system by antitumor 2-imidazolyl disulfides.

The interactions of a series of 2-imidazolyl disulfide antitumor compounds with the thioredoxin reductase(TR)/thioredoxin (hTrx) redox system have been studied. Disulfides III-2 (n-butyl 2-mercaptoimidazolyl disulfide) and VI-2 (ethyl 2-mercaptoimidazolyl disulfide) were substrates for reduction by TR with Km values of 43 and 48 microM. Disulfides IV-2 (1-methylpropyl 2-mercaptoimidazolyl disulfide) and DLK-36 (benzyl 2-mercaptoimidazolyl disulfide) were competitive inhibitors of the reduction of hTrx by TR with Ki values of 31 microM. None of the disulfides were substrates for reduction by human glutathione reductase. The disulfides caused reversible thioalkylation of hTrx at the redox catalytic site as shown by the fact that there was no thioalkylation of a mutant hTrx where both the catalytic site Cys32 and Cys35 residues were replaced by Ser. In addition, the disulfides caused a slower irreversible inactivation of hTrx as a substrate for reduction by TR, with half-lives for III-2 of 30 min, for IV-2 of 4 hr, and for IX-2 (t-butyl 2-mercaptoimidazolyl disulfide) of 24 hr. This irreversible inactivation of hTrx occurred at concentrations of the disulfides an order of magnitude below those that inhibited TR, and involved the Cys73 of hTrx, which is outside the conserved redox catalytic site, as shown by the resistance to inactivation of a mutant hTrx where Cys73 was replaced by Ser. Electrophoretic and mass spectral analyses of the products of the reaction between the disulfides and hTrx show that modification of 1-3 Cys residues of the protein occurred in a concentration-dependent fashion. The disulfides inhibited the hTrx-dependent proliferation of MCF-7 breast cancer cells with IC50 values for III-2 and IV-2 of 0.2 and 1.2 microM, respectively. The results show that although the catalytic sites of TR and hTrx are reversibly inhibited by the 2-imidazolyl disulfides, it is the irreversible thioalkylation of Cys73 of hTrx by the disulfides that most probably accounts for the inhibition of thioredoxin-dependent cell growth by the disulfides.

Study of non-covalent enzyme-inhibitor complexes of aldose reductase by electrospray mass spectrometry.

Specific non-covalent interactions between aldose reductase (AR), its NADP+ cofactor and five inhibitors have been characterized by electrospray mass spectrometry (ES-MS). These results indicated that the protein could be desorbed and maintained in the gas phase in a form very close to its native conformation. Collisionally induced dissociation (CID)-MS and CID-MS-MS showed that the adenosine diphosphate part of the cofactor interacts strongly with AR. The relative stability of the ternary AR x NADP+ x inhibitor complexes was established and successfully correlated with the IC50 values. All inhibitors were shown to only bind to AR holoenzyme. These results are important for the field of drug development insofar as ES-MS might provide a rapid and very sensitive method for the screening of potential drugs or for the identification of compounds displaying high binding affinity to a target biomolecule.

Negative electrospray ionization mass spectrometry of synthetic and chemically modified oligonucleotides.

We report here on the analysis of synthetic oligonucleotides by electrospray ionization mass spectrometry (ESI-MS). After intensive removal of salt ions (especially sodium cations), negative ion mass spectra, allowing mass measurement with an accuracy of 0.01%, were obtained on several oligonucleotides up to 80 nucleotides. In most cases, the resolution was sufficient to observe n-1 and n-2 forms due to internal deletions during automated synthesis, and to identify the missing nucleotides. A 132-mer, whose size is close to the limit of automated chemical synthesis, was also successfully mass measured. A quantitative study showed that ESI-MS can provide quantitative data on oligonucleotides of similar size and structure. The described methodology is used to characterize oligonucleotide analogues such as phosphorothioate oligonucleotides designed for antisense applications. Finally, analyses in the positive ion mode on a trimer TpTpT in the presence of different amine bases were performed and allowed a better understanding of the influence of these bases on the ions formation.

Sequence of pig lens aldose reductase and electrospray mass spectrometry of non-covalent and covalent complexes.

The complete sequence of pig lens aldose reductase (EC 1.1.1.21), a member of the nicotinamide coenzyme-dependent aldo-keto reductase super family, was determined by the combined use of data obtained from Edman degradation, fast-atom-bombardment mass spectrometry and electrospray mass spectrometry. The N-terminal residue of human and pig aldose reductase was shown to be acetylated. The assignment of a disulfide bridge (Cys298-Cys303) was obtained by mass spectrometry. Electrospray mass spectrometry has been used for molecular mass measurement of human muscle (35758 +/- 7 Da) and pig lens (35778 +/- 3Da) aldose reductase; using mild ionization conditions, it has also been used to study the reversible interaction involved in a non-covalent complex with NADP+ (36527 +/- 4Da). An alkylating analog of NADP+ (3-chloroacetylpyridine-adenine dinucleotide phosphate) was used as an irreversible inhibitor to investigate the NADP binding site and the mass of the covalent complex was measured (36521 +/- 3 Da).