Can immunoglobulin C(H)1 constant region domain modulate antigen binding affinity of antibodies?

Although the switch process is frequently associated with affinity maturation, the constant region is not assumed to play a role in Ag-Ab binding. In the present work, we demonstrate that two clonally related human monoclonal Igs sharing identical V(H) and V(L) sequences, but expressing different isotypes (IgA1kappa(PER) and IgG1kappa(PER)), bind tubulin with significantly different affinities. This difference was mainly accounted for by a disparity in the association rate constants. These results suggest that affinity maturation of this clone could be achieved through class switching in the absence of further somatic mutations. Since the differences observed were found at the Fab level, they also suggest a role for the C(H)1 domain in structuring the Ag-binding site into a more kinetically competent form.

Further characterization of the two tetraheme cytochromes c3 from Desulfovibiro africanus: nucleotide sequences, EPR spectroscopy and biological activity.

The genes encoding the basic and acidic tetraheme cytochromes c3 from Desulfovibrio africanus have been sequenced. The corresponding amino acid sequences of the basic and acidic cytochromes c3 indicate that the mature proteins consist of a single polypeptide chain of 117 and 103 residues, respectively. Their molecular masses, 15102 and 13742 Da, respectively, determined by mass spectrometry, are in perfect agreement with those calculated from their amino acid sequences. Both D. africanus cytochromes c3 are synthesized as precursor proteins with signal peptides of 23 and 24 residues for the basic and acidic cytochromes, respectively. These cytochromes c3 exhibit the main structural features of the cytochrome c3 family and contain the 16 strictly conserved cysteine + histidine residues directly involved in the heme binding sites. The D. africanus acidic cytochrome c3 differs from all the other homologous cytochromes by its low content of basic residues and its distribution of charged residues in the amino acid sequence. The presence of four hemes per molecule was confirmed by EPR spectroscopy in both cytochromes c3. The g-value analysis suggests that in both cytochromes, the angle between imidazole planes of the axial histidine ligands is close to 90 degrees for one heme and much lower for the three others. Moreover, an unusually high exchange interaction (approximately 10[-2] cm[-1]) was evidenced between the highest potential heme (-90 mV) and one of the low potential hemes in the basic cytochrome c3. The reactivity of D. africanus cytochromes c3 with heterologous [NiFe] and [Fe] hydrogenases was investigated. Only the basic one interacts with the two types of hydrogenase to achieve efficient electron transfer, whereas the acidic cytochrome c3 exchanges electrons specifically with the basic cytochrome c3. The difference in the specificity of the two D. africanus cytochromes c3 has been correlated with their highly different content of basic and acidic residues.

MCD and 1H-NMR spectroscopic studies of Desulfovibrio africanus ferredoxin I: revised amino-acid sequence and identification of secondary structure.

Desulfovibrio africanus ferredoxin I was studied by magnetic circular dichroism and 1H-NMR spectroscopies. These showed the presence of histidine and tryptophan, in contrast to the previously reported amino-acid sequence (Bruschi and Hatchikian (1982) Biochimie 64, 503-507). This was redetermined and the revised sequence shown to contain both histidine and tryptophan, as well as four other corrections (Sery et al. (1994) Biochemistry, submitted). Electrospray mass spectrometry confirmed the mass of the ferredoxin was that given by the revised amino-acid sequence. The secondary structure of the ferredoxin I was investigated with two-dimensional 1H-NMR experiments and both alpha-helix and beta-sheet structure detected. The influence of the paramagnetism of the Fe4 S4 cluster on the NMR properties of the ferredoxin protons was investigated, by temperature-dependent experiments, and it was concluded that there is only a negligible dipolar contribution to resonance chemical shifts from this source. The significance of this for the determination of the three-dimensional structure of the ferredoxin by NMR is discussed.

The irreversible inactivation of ribonucleotide reductase from Escherichia coli by superoxide radicals.

The expression of superoxide dismutase in all aerobic living organisms supports the concept that superoxide radicals are toxic species. However, because of the limited chemical reactivity of superoxide, the mechanisms of this toxicity are still uncertain. Protein R2, the small component of ribonucleotide reductase, a key enzyme for DNA synthesis, is shown here to be irreversibly inactivated during incubation with an enzymatic generator of superoxide radicals, at neutral pH. During inactivation the essential tyrosyl radical of protein R2 is irreversibly destroyed. Full protection is afforded by superoxide dismutase. It is proposed that coupling between superoxide radicals and the radical protein R2 generates oxidized forms of tyrosine, tyrosine peroxide and 3,4-dihydroxyphenylalanine.

Identification of a cryptic protein kinase CK2 phosphorylation site in human complement protease Clr, and its use to probe intramolecular interaction.

Treatment of human (activated)C1r by CK2 resulted in the incorporation of [32P]phosphate into the N-terminal alpha region of its non-catalytic A chain. Fragmentation of 32P-labelled (activated)C1r followed by N-terminal sequence and mass spectrometry analyses allowed identification of Ser189 as the phosphorylation site. Accessibility of Ser189 was low in intact C1r, due in part to the presence of one of the oligosaccharides borne by the alpha region, further reduced in the presence of calcium, and abolished when C1r was incorporated into the C1s-C1r-C1r-C1s tetramer or the C1 complex. In contrast, phosphorylation was enhanced in the isolated alpha fragment and insensitive to calcium. Taken together, these data provide support for the occurrence of a (Ca2+)-dependent interaction between the alpha region and the remainder of the C1r molecule.

Analysis of the N-linked oligosaccharides of human C1s using electrospray ionisation mass spectrometry.

Information on the structures of the oligosaccharides linked to Asn residues 159 and 391 of the human complement protease C1s was obtained using mass spectrometric and monosaccharide analyses. Asn159 is linked to a complex-type biantennary, bisialylated oligosaccharide NeuAc2 Gal2 GlcNAc4 Man3 (molecular mass = 2206 +/- 1). Asn391 is occupied by either a biantennary, bisialylated oligosaccharide, or a triantennary, trisialylated species NeuAc3 Gal3 GlcNAc5 Man3 (molecular mass = 2861 +/- 1), or a fucosylated triatennary, trisialylated species NeuAc3 Gal3 GlcNAc5 Man3 Fuc1 (molecular mass = 3007 +/- 1), in relative proportions of approximately 1:1:1. The carbohydrate heterogeneity at Asn391 gives rise to three major types of C1s molecules of molecular masses 79,318 +/- 8 (A), 79,971 +/- 8 (B), and 80,131 +/- 8 (C), with an average mass of 79,807 +/- 8. A minor modification, yielding an extra mass of 132 +/- 2, is also detected within positions 1-153.

The protein sequence of an archaeal catalase-peroxidase.

The gene encoding a catalase-peroxidase of archaeal origin, the halophilic catalase-peroxidase from Haloarcula marismortui, was sequenced. The primary structure proposed was confirmed by Edman degradation and mass spectrometry analyses of proteolytic fragments of the purified protein. The open reading frame in the gene corresponds to 731 amino acids and the calculated mass of the mature protein (deleted of the N-terminal methionine) is 81,253.65 Da, in reasonable agreement with the value of 81,292 +/- 9 Da previously measured by mass spectrometry. Southern and Northern blot analyses showed that the protein is encoded by a single gene as a monocistronic transcript. The protein sequence shows a high level of identity with bacterial catalase-peroxidases, with strongly conserved regions around the heme binding histidines. Similarly to other soluble halophilic proteins, it shows the excess of acidic residues that has been associated with solvation in halophilic adaptation.

Polypeptide:N-acetylgalactosaminyltransferase activities towards the mucin MUC5AC peptide motif using microsomal preparations of normal and tumoral digestive mucosa.

The selected-acceptor substrate peptide (TTSAPTTS), deduced from the human mucin gene MUC5AC (expressed essentially in the human gastric and tracheobronchial mucosa), was used to assay polypeptide:N-acetylgalactosaminyltransferases (GalNAc transferases) of different microsomal preparations, obtained from gastric and colonic mucosa in normal and tumoral situations. The O-glycosylated products, analyzed by capillary electrophoresis and electrospray mass spectrometry, showed a variable number of GalNAc O-linked to the different hydroxy amino acids of TTSAPTTS, depending on the tissue studied. Our observations were consistent with the existence of more than one form of GalNAc transferases which were expressed differentially in the gastrointestinal tract (stomach and/or colon). The levels of enzyme activities showed a tissue-specific pattern as they were high in normal colonic tissue and low in colon cancer. On the other hand, in the tumoral gastric tissue (displaying intestinal metaplasia) a high level of GalNAc transferase activities was obtained, similar to that found in the normal colon. Moreover, slight discrepancies (activities and number of O-linked GalNAc) were only detected between normal gastric and tumoral colonic preparations. Thus, the data indicated that the dedifferentiation of the gastric cancer tissue may induce GalNAc transferase activities similar to those in the normal colonic, tissue and that colonic and gastric tissues may contain families of glycosyltransferases involved specifically in reaction towards particular peptide or protein substrates. In addition, the analysis by capillary electrophoresis and electrospray mass spectrometry revealed, in tumoral gastric as well as in normal colonic tissues, a high dipeptidylaminotransferase activity inducing an elongation of TTSAPTTS by dithreonine. This activity was low in normal gastric and tumoral colonic tissues.

Multi-compartment heart segmentation in CT angiography using a spatially varying gaussian classifier.

OBJECTIVE: A fully automated and efficient method for segmenting ten major structures within the heart in Cardiac CT Angiography data for the purposes of display or cardiac functional analysis. MATERIALS AND METHODS: A spatially varying Gaussian classifier is a flexible model for segmentation, combining the advantages of atlas-based frameworks, with supervised intensity models. It is composed of an independent Gaussian classifier at each voxel and uses non-rigid registration for the initial spatial alignment. We show how this large model can be trained efficiently and present a novel smoothing technique based on normalised convolution to mitigate inherent overfitting issues. The 30 datasets used in this study are selected from a variety of different scanners in order to test the robustness and stability of the algorithm. The datasets were manually segmented by a trained clinician. RESULTS: The method was evaluated in a leave-one-out fashion, and the results were compared to other state of the art methods in the field, with a mean surface-to-surface distance of between 0.61 and 2.12 mm for different compartments. CONCLUSION: The accuracy of this method is comparable to other state of the art methods in the field. Its benefits lie in its conceptual simplicity and its general applicability. Only one non-rigid registration is required, giving it a speed advantage over multi-atlas approaches. Further accuracy may be achievable through the incorporation of an explicit shape model.

Electrospray-ionization mass spectrometry of molecular variants of a [2Fe-2S] ferredoxin.

The [2Fe-2S] ferredoxin from Clostridium pasteurianum is a homodimeric protein of which each subunit contains one [2Fe-2S] cluster. In previous investigations, the five cysteine residues in positions 11, 14, 24, 56 and 60 had been mutated into serine or alanine. The wild type ferredoxin and several of its molecular variants have now been analyzed by electrospray-ionization mass spectrometry. In the negative-ion detection mode, depending on the infusion solvent used, molecular peaks attributable to the apoprotein, to the monomeric holoprotein, and to the dimeric holoprotein were detected in all cases. The data confirmed the presence of the expected mutations, showed that all of these proteins contain one [2Fe-2S] cluster per subunit, and indicated that the dimeric structure of these ferredoxins could be retained in the conditions of the electrospray ionization. This investigation establishes the power of electrospray-ionization mass spectrometry for the analysis of oligomeric proteins containing labile metal clusters.

Observation of holoprotein molecular ions of several ferredoxins by electrospray-ionization-mass spectrometry.

Several ferredoxins containing [4Fe-4S] or [2Fe-2S] active sites have been analyzed by electrospray-ionization-mass spectrometry. For these acidic proteins, low pH conditions must be implemented in order to ensure strong signals in positive-ionization mode. Under such conditions the iron-sulfur active sites were lost in most cases. In contrast, the holoproteins were preserved under negative-ionization mode conditions: they were weakly but sufficiently ionized and information about their cofactor content could be obtained. The experimental conditions set up here should provide a useful basis for the detailed characterization of more complex iron-sulfur proteins.

Deacylation kinetics analysis of Streptococcus pneumoniae penicillin-binding protein 2x mutants resistant to beta-lactam antibiotics using electrospray ionization- mass spectrometry.

Penicillin-binding proteins (PBPs) catalyze the transpeptidase reaction involved in peptidoglycan synthesis and are covalently inhibited by the beta-lactam antibiotics. In a previous work we have focused on acylation efficiency measurements of various Streptococcus pneumoniae PBP2x* mutants to study the molecular determinants of resistance to beta-lactams. In the present paper we have developed a method to improve an accurate determination of the deacylation rate constant using electrospray ionization-mass spectrometry. This method is adaptable to the analysis of deacylation of any beta-lactam. Compared to the fluorographic technique, the ESI-MS method is insensitive to variations in the concentration of functional proteins and is therefore more reliable. We have established that the resistance of PBPs to beta-lactams is mostly due to a decrease of the acylation efficiency with only marginal effects on the deacylation rates.

Primary structure of Chromatium tepidum high-potential iron-sulfur protein in relation to thermal denaturation.

A high-potential ferredoxin (HiPIP) has been purified from the thermophilic purple sulfur bacterium Chromatium tepidum. Most of the properties of this protein, including absorption and electron paramagnetic resonance spectra as well as redox potential, are identical to those of the similar protein isolated from the mesophilic organism Chromatium vinosum. The similarity extends to the amino acid sequences, which share 74 of the 83 residues composing the primary structure of C. tepidum HiPIP. The latter has been determined by sequencing overlapping peptides and precisely measuring the molecular mass of the holoprotein (9136 Da) by electrospray ionization mass spectrometry. The most significant difference between these sequences involves a stretch of 8 amino acids, which is shortened by two residues and notably changed in C. tepidum HiPIP. This region had been identified in the three-dimensional structure of C. vinosum HiPIP as both a link between two strands of a twisted beta sheet coordinating the [4Fe-4S] cluster and an area of strong interaction of the molecule with the solvent. These data have been used to discuss the molecular basis for the slightly improved thermal stability of C. tepidum HiPIP, as compared to C. vinosum HiPIP. Based on the physiological differences distinguishing C. tepidum from other small-sized Chromatiaceae, the presence of an abundant HiPIP in C. tepidum indicates that involvement as electron acceptor for the previously proposed thiosulfate oxidizing activity in C. vinosum may not be the sole function in all purple sulfur bacteria.

Analysis, by electrospray ionization mass spectrometry, of several forms of Clostridium pasteurianum rubredoxin.

Clostridium pasteurianum rubredoxin and its recombinant counterpart purified from Escherichia coli have been analysed by electrospray ionization m.s. (e.s.i.m.s.). Whereas the N-terminal methionine of the native protein is formylated, the recombinant one has a free N-terminal methionine. E. coli cells also produce a colourless protein from the cloned gene. This protein is absent from C. pasteurianum and was shown to be zinc-substituted rubredoxin. The molecular forms of rubredoxin detected by e.s.i.m.s. depended on the experimental conditions used. Significant conversion into apo-rubredoxin occurred when the proteins were ionized at acidic pH and detected in the positive-ion mode. This conversion was quantitative in the case of Zn-rubredoxin. In contrast, when the proteins were analysed at neutral pH in the negative-ion mode, only the holoproteins, i.e. the species initially present in the solutions, were detected in the spectra. The e.s.i.m.s. experimental conditions set up here may prove useful for the analysis of other acidic metalloproteins with weakly bound metals.

Determination of the C-terminal form of an anemone toxin using capillary electrophoresis and mass spectrometry.

To identify the form of the C-terminal amino acid of a sea anemone toxin, the native protein was compared with two synthetic proteins comprising the same sequence and a free or an amide C-terminal form. Using electrospray ionization-mass spectrometry, capillary electrophoresis and the coupling of both techniques, we assigned the C-terminus of the native protein to be in the free carboxyl form.

Use of a designed fusion protein dissociates allosteric properties from the dodecameric state of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase.

The catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa, an enzyme consisting of 12 identical 38-kDa subunits, displays allosteric properties, namely carbamoylphosphate homotropic cooperativity and heterotropic activation by AMP and other nucleoside monophosphates and inhibition by polyamines. To shed light on the effect of the oligomeric organization on the enzyme's activity and/or allosteric behavior, a hybrid ornithine carbamoyltransferase/glutathione S-transferase (OTCase-GST) molecule was constructed by fusing the 3' end of the P. aeruginosa arcB gene (OTCase) to the 5' end of the cDNA encoding Musca domestica GST by using a polyglycine encoding sequence as a linker. The fusion protein was overexpressed in Escherichia coli and purified from cell extracts by affinity chromatography, making use of the GST domain. It was found to exist as a trimer and to retain both the homotropic and heterotropic characteristic interactions of the wild-type catabolic OTCase but to a lower extent as compared with the wild-type OTCase. The dodecameric organization of catabolic P. aeruginosa OTCase may therefore be related to an enhancement of the substrate cooperativity already present in its trimers (and perhaps also to the thermostability of the enzyme).

Biochemical and spectroscopic characterization of two new cytochromes isolated from Desulfuromonas acetoxidans.

The multimeric cytochromes described to date in sulfate- and sulfur-reducing bacteria are associated with diverse respiratory modes involving the use of elemental sulfur or oxidized sulfur compounds as terminal acceptors. They exhibit no structural similarity with the other cytochrome c classes and are characterized by a bis-histidinyl axial iron coordination and low redox potentials. We have purified two new cytochromes c with markedly different molecular masses (10 000 and 50 000) from the bacterium Desulfuromonas acetoxidans, which uses anaerobic sulfur respiration as its sole energy source. The characterization by electrochemistry and optical and EPR spectroscopies revealed the cytochrome c (Mr = 10 000) to be the first monohemic cytochrome c exhibiting a bis-histidinyl axial coordination and a low redox potential (-220 mV). The cytochrome c (Mr = 50 000) contains four hemes of low potential (-200, -210, -370, and -380 mV) with the same axial coordination. The N-terminal amino acid sequences were compared with that of the trihemic cytochrome c7, previously described in D. acetoxidans and which is related to tetrahemic cytochrome c3 from sulfate reducing bacteria. Some homology was found between cytochrome c (Mr = 10 000) and cytochrome c7. Both D. acetoxidans cytochromes c are located in the periplasmic space and their biochemical and spectroscopic properties indicate that they belong to the class III cytochromes.

Structural and kinetic studies of the pyruvate-ferredoxin oxidoreductase/ferredoxin complex from Desulfovibrio africanus.

The pyruvate-ferredoxin oxidoreductase (PFOR)/ferredoxin (Fd) system of Desulfovibrio africanus has been investigated with the aim of understanding more fully protein-protein interaction and the kinetic characteristics of electron transfer between the two redox partners. D. africanus contains three Fds (Fd I, Fd II and Fd III) able to function as electron acceptors for PFOR. The complete amino acid sequence of Fd II was determined by automatic Edman degradation. It revealed a striking similarity to that of Fd I. The protein consists of 64 residues and its amino acid sequence is in agreement with a molecular mass of 6822.5 Da as measured by electrospray MS. Fd II contains five cysteine residues of which the first four (Cys11, Cys14, Cys17 and Cys54) are likely ligands for the single [4Fe-4S] cluster. A covalently cross-linked complex between PFOR and Fd I or Fd II was obtained by using a water soluble carbodiimide. This complex exhibited a stoichiometry of one ferredoxin for one PFOR subunit and is dependent on the ionic strength. The second-order rate constants for electron transfer between PFOR and Fds determined electrochemically using cyclic voltammetry are 7 x 107 M-1.s-1 for Fd I and 2 x 107 M-1.s-1 for Fd II and Fd III. The Km values of PFOR for Fd I and Fd II measured both by the electrochemical and the spectrophotometric method have been found to be 3 microM and 5 microM, respectively. The three-dimensional modelling of Fd II and surface analysis of Fd I, Fd II and PFOR suggest that a protein-protein complex is likely to be formed between aspartic acid/glutamic acid invariant residues of Fds and lysine residues surrounding the distal [4Fe-4S] cluster of PFOR. All of these studies are indicative of the involvement of electrostatic interactions between the two redox partners.

A [2Fe-2S] protein from the hyperthermophilic bacterium Aquifex aeolicus.

Overexpression in Escherichia coli of the fdx4 gene from Aquifex aeolicus has allowed isolation and characterization of the first hyperthermophilic [2Fe-2S](Scys)(4) protein, a homodimer of M = 2 x 12.4 kDa with one [2Fe-2S] cluster per subunit. This protein is undamaged by heating to 100 degrees C for at least three hours. The primary structure, in particular the characteristic distribution of the four cysteine ligands of the metal site, and the spectroscopic properties of the A. aeolicus protein relate it to well characterized [2Fe-2S] proteins from Clostridium pasteurianum and Azotobacter vinelandii. These proteins are also homologous to subunits or domains of hydrogenases and NADH-ubiquinone oxidoreductase (Complex I) of respiratory chains. The A. aeolicus [2Fe-2S] protein is thus representative of a presumably novel protein fold involved in a variety of functions in very diverse cellular backgrounds.