Fluorimetric screening assay for protein carbonyl evaluation in biological samples.

Many assays are available for the detection of protein carbonyls (PCs). Currently, the measurement of PC groups after their derivatization with 2,4-dinitrophenol hydrazine (DNPH) is widely used for measuring protein oxidation in biological samples. However, this method includes several washing steps. In this context, we have developed a rapid, sensitive, and accurate fluorimetric method adapted to 96-well microplates for the convenient assessment of protein carbonyl level in biological samples. The method reported here is based on the reaction of carbonyl content in proteins with 7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole (NBDH) to form highly fluorescent derivatives via hydrazone formation. PCs were determined using the DNPH and NBDH assays in fully reduced bovine serum albumin (BSA) and plasma and liver homogenates obtained from healthy control rats up the addition of various amounts of HOCl-oxidized BSA (OxBSA). Using the NBDH assay, PC concentrations as low as 0.2 nmol/mg were detected with precision as low as 5%. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy was used to successfully identify the formation of the NBDH adducts after derivatization with standard oxidized peptides. Finally, the two methods were further used for PC determination in plasma and liver samples from diabetic and normal rats, showing that the NBDH assay can be reliably used in biological experiments.

[Thermodynamics of calmodulin and tubulin binding to the vinca-alkaloid vinorelbine].

Vinca-alkaloids, such as vinblastine, and some of their derivatives, as for example vinorelbine, are widely used in clinical therapy of leukemia and several types of tumors. Their effects are associated with the disfunctioning of the mitotic spindle, which leads to mitosis blockage and a shutdown of the cell cycle. Their primary target is tubulin, however recent research has shown that some of the vinca-alkaloids inhibit calmodulin binding to its targets. Vinka-alkaloids binding with other proteins could be responsible for their efficiency and neuroprotection. Here we investigated the thermodynamics of vinorelbine interactions with calmodulin and tubulin. It was determined that unlike the other vinca-alkaloids both vinorelbine binding sites are located in the C-domain of calmodulin, and characterized by association constants of 4.0 x 10(5) and 5.4 x 10(4) M(-1). At the same time the thermodynamics of vinorelbine binding to tubulin are not much different from that of other vinca-alkaloids. These results will allow getting a better insight on the reaction mechanisms of vinca-alkaloids on a secondary protein target.

Detection of bile salt-dependent lipase, a 110 kDa pancreatic protein, in urines of healthy subjects.

Bile salt-dependent lipase (BSDL), a 110 kDa glycoprotein secreted by the pancreatic acinar cells, participates in the duodenal hydrolysis of dietary lipid esters. Recent in vitro and in vivo studies demonstrated that the BSDL reaches the blood via a transcytosis motion through enterocytes, suggesting that this enzyme may play a role in vascular biology. Once in the blood, BSDL should be eliminated. We address the hypothesis that BSDL may be filtered by the glomerulus and eliminated in urines. Immunological methods and proteomic were used to detect and to characterize BSDL in urine. The immunoreactive form of BSDL was detected in urines of 36 male subjects devoid of renal failure. Proteomic demonstrated that the immunoreactive protein is BSDL. Experiments using a monoclonal antibody to the oncofetal glycoform of pancreatic BSDL suggested that the protein is not expressed by renal cells but originates from the pancreas via circulation. We demonstrate that under normal physiological conditions, BSDL, a high-molecular weight blood glycoprotein, can be filtered by the renal glomerulus to be eliminated in urines.

Protein-ligand and protein-protein interactions studied by electrospray ionization and mass spectrometry.

Electrospray ionization has made possible the transference of non-covalently bound complexes from solution phase to high vacuum. In the process, a complex acquires a net charge and becomes amenable to measurement by MS. FTICR (Fourier-transform ion cyclotron resonance) MS allows these ions to be measured with sufficiently high resolution for the isotopomers of complexes of small proteins to be resolved from each other (true for complexes up to about 100 kDa for the most powerful FTICR instruments), which is of crucial significance in the interpretation of spectra. Results are presented for members of the S100 family of proteins, demonstrating how non-covalently bound complexes can be distinguished unambiguously from covalently bound species. Consideration relevant both to determination of binding constants in solution from the gas-phase results and to the elucidation of protein folding and unfolding in solution are discussed. The caveats inherent to the basic approach of using electrospray and MS to characterize protein complexes are weighed and evaluated.

Repair of oxidized proteins. Identification of a new methionine sulfoxide reductase.

Oxidation of methionine residues to methionine sulfoxide can lead to inactivation of proteins. Methionine sulfoxide reductase (MsrA) has been known for a long time, and its repairing function well characterized. Here we identify a new methionine sulfoxide reductase, which we referred to as MsrB, the gene of which is present in genomes of eubacteria, archaebacteria, and eucaryotes. The msrA and msrB genes exhibit no sequence similarity and, in some genomes, are fused. The Escherichia coli MsrB protein (currently predicted to be encoded by an open reading frame of unknown function named yeaA) was used for genetic, enzymatic, and mass spectrometric investigations. Our in vivo study revealed that msrB is required for cadmium resistance of E. coli, a carcinogenic compound that induces oxidative stress. Our in vitro studies, showed that (i) MsrB and MsrA enzymes reduce free methionine sulfoxide with turn-over rates of 0.6 min(-1) and 20 min(-1), respectively, (ii) MsrA and MsrB act on oxidized calmodulin, each by repairing four to six of the eight methionine sulfoxide residues initially present, and (iii) simultaneous action of both MsrA and MsrB allowed full reduction of oxidized calmodulin. A possibility is that these two ubiquitous methionine sulfoxide reductases exhibit different substrate specificity.

Phosphorylation and oligomerization states of native pig brain HSP90 studied by mass spectrometry.

HSP90 is one of the most abundant proteins in the cytosol of eukaryotic cells. HSP90 forms transient or stable complexes with several key proteins involved in signal transduction including protooncogenic protein kinases and nuclear receptors, it interacts with cellular structural elements such as actin-microfilament, tubulin-microtubule and intermediate filaments, and also exhibits conventional chaperone functions. This protein exists in two isoforms alpha-HSP90 and beta-HSP90, and it forms dimers which are crucial species for its biological activity. PAGE, ESI-MS and MALDI-MS were used to study HSP90 purified from pig brain. The two protein isoforms were clearly distinguished by ESI-MS, the alpha isoform being approximately six times more abundant than the beta isoform. ESI-MS in combination with lambda phosphatase treatment provided direct evidence of the existence of four phosphorylated forms of native pig brain alpha-HSP90, with the diphosphorylated form being the most abundant. For the beta isoform, the di-phosphorylated was also the most abundant. MALDI mass spectra of HSP90 samples after chemical cross-linking showed a high percentage of alpha-alpha homodimers. In addition, evidence for the existence of higher HSP90 oligomers was obtained.

The dimerization interface of the metastasis-associated protein S100A4 (Mts1): in vivo and in vitro studies.

The S100 calcium-binding proteins are implicated in signal transduction, motility, and cytoskeletal dynamics. The three-dimensional structure of several S100 proteins revealed that the proteins form non-covalent dimers. However, the mechanism of the S100 dimerization is still obscure. In this study we characterized the dimerization of S100A4 (also named Mts1) in vitro and in vivo. Analytical ultracentrifugation revealed that apoS100A4 was present in solution as a mixture of monomers and dimers in a rapidly reversible equilibrium (K(d) = 4 +/- 2 microm). The binding of calcium promoted dimerization. Replacement of Tyr-75 by Phe resulted in the stabilization of the dimer. Helix IV is known to form the major part of the dimerization interface in homologous S100 proteins. By using the yeast two-hybrid system we showed that only a few residues of helix IV, namely Phe-72, Tyr-75, Phe-78, and Leu-79, are essential for dimerization in vivo. A homology model demonstrated that these residues form a hydrophobic cluster on helix IV. Their role is to stabilize the structure of individual subunits rather than provide specific interactions across the dimerization surface. Our mutation data showed that the specificity at the dimerization surface is not particularly stringent, which is consistent with recent data indicating that S100 proteins can form heterodimers.

Heterocomplex formation between metastasis-related protein S100A4 (Mts1) and S100A1 as revealed by the yeast two-hybrid system.

S100A4 (Mts1) is a Ca(2+)-binding protein of the S100 family. This protein plays an important role in promoting tumor metastasis. In order to identify S100A4 interacting proteins, we have applied the yeast two-hybrid system as an in vivo approach. By screening a mouse mammary adenocarcinoma library, we have demonstrated that S100A4 forms a heterocomplex with S100A1, another member of the S100 family. The non-covalent heterodimerization was confirmed by fluorescence spectroscopy and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Mutational analysis revealed that replacement of Cys(76) and/or Cys(81) of S100A4 by Ser abolishes the S100A4/S100A1 heterodimerization, but does not affect the S100A4 homodimerization in vivo.

Calmodulin-peptide interactions: apocalmodulin binding to the myosin light chain kinase target-site.

Noncovalent binding of the synthetic peptide RS20 to calmodulin in the presence of calcium was confirmed by electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry to form a complex with a 1:1:4 calmodulin/RS20/calcium stoichiometry. There was no evidence for formation of a calmodulin-RS20-Ca(2) species. The absence of calmodulin-RS20-Ca(2) would be consistent with models in which the two globular domains are coupled functionally. There was evidence that calmodulin, RS20-calmodulin without associated calcium, and calmodulin-RS20-Ca(4) existed together in solution, whereas calmodulin-calcium complexes were absent. It is proposed that calcium binding to form the calmodulin-RS20-Ca(4) complex occurs after an initial RS20-calmodulin binding event, and serves to secure the target within the calmodulin structure. The binding of more than one RS20 molecule to calmodulin was observed to induce unfolding of calmodulin.

A novel Ca2+ binding protein associated with caldesmon in Ca2+-regulated smooth muscle thin filaments: evidence for a structurally altered form of calmodulin.

Smooth muscle thin filaments are made up of actin, tropomyosin, the inhibitory protein caldesmon and a Ca2+-binding protein. Thin filament activation of myosin MgATPase is Ca2+-regulated but thin filaments assembled from smooth muscle actin, tropomyosin and caldesmon plus brain or aorta calmodulin are not Ca2+-regulated at 25 degrees C/50 mM KCl. We isolated the Ca2+-binding protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chromatography in 6 M urea and phenyl sepharose chromatography using sheep aorta as our starting material. CaBP combines with smooth muscle actin, tropomyosin and caldesmon to reconstitute a normally regulated thin filament at 25 degrees C/50 mM KCl. It reverses caldesmon inhibition at pCa5 under conditions where CaM is largely inactive, it binds to caldesmon when complexed with actin and tropomyosin rather than displacing it and it binds to caldesmon independently of [Ca2+]. Amino acid sequencing, and electrospray mass spectrometry show the CaBP is identical to CaM. Structural probes indicate it is different: calmodulin increases caldesmon tryptophan fluorescence but CaBP does not. The distribution of charged species in electrospray mass spectrometry and nozzle skimmer fragmentation patterns are different indicating a less stable N-terminal lobe for CaBP. Brief heating abolishes these special properties of the CaBP. Mass spectrometry in aqueous buffer showed no evidence for the presence of any covalent or non-covalently bound adduct. The only remaining conclusion is that CaBP is calmodulin locked in a metastable altered state.

Apocalmodulin binds to the myosin light chain kinase calmodulin target site.

The interaction of a 20-residue-long peptide derived from the calmodulin-binding domain of the smooth muscle myosin light chain kinase with calcium-free calmodulin (apocalmodulin) was studied using a combination of isothermal titration calorimetry and differential scanning calorimetry. We showed that: (i) a significant binding between apocalmodulin and the target peptide (RS20) exists in the absence of salt (Ka = 10(6) M-1), (ii) the peptide interacts with the C-terminal lobe of calmodulin and adopts a partly helical conformation, and (iii) the presence of salt weakens the affinity of the peptide for apocalmodulin, emphasizing the importance of electrostatic interactions in the complex. Based on these results and taking into account the work of Bayley et al. (Bayley, P. M., Findlay, W.A., and Martin, S. R. (1996) Protein Sci. 5, 1215-1228), we suggest a physiological role for apocalmodulin.

Evidence of noncovalent dimerization of calmodulin.

Calcium-binding proteins, such as S-100, dimerize readily, and this phenomenon plays an important role in their regulation of target enzymes [Krebs, J., Quadroni, M. & Van Eldik, L.J. (1995) Nat. Struct. Biol. 2, 711-714; Kilby, P.M., Van Eldik, L.J. & Roberts, G. C. (1996) Structure 4, 1041-1052]. We have investigated by Fourier-transform ion cyclotron resonance (FTICR) MS the conformational states of the calcium-binding protein calmodulin, and present clear evidence for a calmodulin dimer formed as a result of noncovalent interactions between folded monomers. Ultra-high-resolution electrospray ionization (ESI) mass spectra for calmodulin, obtained with a 9.4 T FTICR mass spectrometer, are presented. With the use of denaturing solutions (1 : 1 acetonitrile/water + 1% formic acid), relatively high charge states (20 < z < 10) of monomeric calmodulin ions were detected, whereas when calmodulin was electrosprayed from buffer, monomers ions with only 5-10 charges were detected. CD measurements for calmodulin in buffered solution revealed that its alpha-helical content was significantly higher than that for calmodulin in acetonitrile/water solutions, consistent with a proposition that changes in charge state distributions observed in the MS experiments reflect differing states of calmodulin folding. Under buffered conditions, noncovalently bound calmodulin dimers were observed by ESI FTICR MS. Analytical ultracentrifugation experiments carried out in the same solution conditions as those used in the MS experiments were consistent with the proposed calmodulin dimer-monomer equilibrium. The ultra-high mass resolution achieved with the 9.4 T FTICR mass spectrometer allowed unequivocal identification of the noncovalent, as opposed to covalent, character of the calmodulin dimer.

Thermodynamic analysis of calcium and magnesium binding to calmodulin.

To elucidate some aspects still debated concerning the interaction of Ca2+ and Mg2+ with CaM, the thermodynamic binding parameters of Ca2+-CaM and Mg2+-CaM complexes were characterized by flow dialysis and isothermal microcalorimetry under different experimental conditions. In particular, the enthalpy and entropy changes associated with Ca2+ and Mg2+ binding to their sites were determined, allowing a better understanding of the mechanism underlying cation-CaM interactions. Ca2+-CaM interaction follows an enthalpy-entropy compensation relationship, suggesting that CaM explores a subspace of isoenergetical conformations which is modified by Ca2+ binding. This Ca2+-induced change in CaM dynamics is proposed to play a key role in CaM function, i.e. in its interaction with and/or activation of target proteins. Furthermore, data show that Mg2+ does not act as a direct competitor for Ca2+ binding on the four main Ca2+ binding sites, but rather as an allosteric effector. This implies that the four main Mg2+ binding sites are distinct from the EF-hand Ca2+ binding sites. Finally, Ca2+ is shown to interact with auxiliary binding sites on CaM. These weak affinity sites were thermodynamically characterized. The results presented here challenge the current accepted view of CaM ion binding.

Conformation and thermal denaturation of apocalmodulin: role of electrostatic mutations.

Scanning microcalorimetry and circular dichroism were used to study conformational state and heat denaturation of Ca2+-free synthetic calmodulin (SynCaM) and three charge reversal mutants. We produced evidence for the major role of the electrostatic potential in the stability and flexibility of SynCaM. The substitution of 118DEE120 by 118KKK120 (SynCaM12A) does not influence the flexibility of the protein; the replacement of 82EEE84 by 82KKK84 (SynCaM8) decreases its level, while the combination of these two mutations in SynCaM18A significantly increases the flexibility. The heat denaturation of apoSynCaM and its mutants is well approximated by two two-state transitions with the lower-temperature transition corresponding to C-terminal lobe melting and the higher-temperature one to N-terminal lobe melting. The difference in transition temperatures for the two lobes decreases in SynCaM8 and increases in SynCaM18A, suggesting a modification in the influence of one lobe to the other. The electrostatic mutations change the parameters of thermal denaturation of SynCaM lobes in a similar way as pH conditions affect thermal transition parameters of multidomain proteins, leading to a linear temperature dependence of transition enthalpy. One domain of the N-terminal lobe in apoSynCaM18A is unfolded in the native state. Near-UV CD spectra point out the invariability of the local structure of aromatic residues upon mutations, although the secondary structure undergoes striking transformations. Cacodylate ions strongly and specifically alter the helical content of SynCaM. Our data unambiguously demonstrate that the two lobes are not independent, and interactions between the lobes are mediated by the electrostatic potential of the molecule.

Analysis of the ion binding sites of calmodulin by electrospray ionization mass spectrometry.

The binding of Ca2+ and Mg2+ to four calmodulins (SynCaM 1, SynCaM 8, SynCaM 12A, and SynCaM 18A) has been studied by ESI-MS. The mass spectra were recorded by dissolving the apoproteins in methanol/water (20/80, v/v) containing 1 mM CaCl2 or 1 mM MgCl2 and the pH adjusted to 6.0 with ammonia. The carrier solvent was methanol/water (20/80, v/v). In the case of Ca2+ complexation, ESI-MS reveals the presence of three kinds of sites: the first of high affinity corresponding to those determined using flow and equilibrium dialysis techniques and two others with lower affinities. These results clearly confirm the conclusion of Milos et al. [Milos, M., Comte, M., Schaer, J. J., & Cox, J. A. (1989) J. Inorg. Biochem. 36, 11-25] that there should exist between four and six auxiliary sites for Ca2+. Concerning the complexation of magnesium, the four proteins are able to bind two Mg2+ almost certainly on auxiliary cationic sites.