RÉSUMÉ
Top-down mass spectrometry has been used to investigate structural diversity within some abundant salivary protein families. In this study, we report the identification of two isoforms of protein II-2 which differed in mass by less than 1 Da, the determination of a sequence for protein IB8a that was best satisfied by including a mutation and a covalent modification in the C-terminal part, and the assignment of a sequence of a previously unreported protein of mass 10433 Da. The final characterization of Peptide P-J was achieved, and the discovery of a truncated form of this peptide was reported. The first sequence assignment was done at low resolution using a hybrid quadrupole time-of-flight instrument to quickly identify and characterize proteins, and data acquisition was switched to Fourier-transform ion cyclotron resonance (FTICR) for proteins that required additional sequence coverage and certainty of assignment. High-resolution and high mass accuracy mass spectrometry on a FTICR-mass spectrometry (MS) instrument combined with electron-capture dissociation (ECD) provided the most informative data sets, with the more frequent presence of "unique" ions that unambiguously define the primary structure. A mixture of predictable and unusual post-translational modifications in the protein sequence precluded the use of shotgun-annotated databases at this stage, requiring manual iterations of sequence refinement in many cases. This led us to propose guidelines for an iterative processing workflow of MS and MSMS data sets that allow researchers to completely assign the identity and the structure of a protein.
Sujet(s)
Protéines/composition chimique , Protéome , Salive/métabolisme , Spectrométrie de masse ESI , Séquence d'acides aminés , Chromatographie en phase liquide à haute performance , Histatines/composition chimique , Humains , Données de séquences moléculairesRÉSUMÉ
Humans deficient in the cerebroside-sulfate activator protein (CSAct or Saposin B) are unable to catabolize sulfatide and other glycosphingolipids leading to their accumulation and neurodegenerative disease. Clinically this usually manifests as a form of metachromatic leukodystrophy (MLD). CSAct is a small water-soluble glycoprotein that apparently functions in the lysosome to solubilize sulfatide and other lipids enabling their interaction with soluble lysosomal hydrolases. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide-sulfate hydrolysis by arylsulfatase A or ex vivo by its ability to functionally complement CSAct deficient fibroblast cell lines derived from MLD patients. A recombinant form of CSAct has been expressed in E. coli and processed in vitro to a form covalently indistinguishable from deglycosylated human CSAct isolated from human urine. Size-exclusion chromatography in combination with multi-angle laser-light scattering (SEC-MALLS) measurements demonstrate that both native and recombinant forms of the molecule behave as a dimer in the pH range 7.0-4.5. The CSAct activity assay showed that both recombinant and deglycosylated human urine CSAct efficiently activated sulfatide sulfate hydrolysis and provided functional complementation of CSAct-deficient cells. However, a D21N mutant form of recombinant CSAct could not functionally complement these cells despite full activity in the in vitro assay. It is concluded that while glycosylation is unnecessary for in vitro and ex vivo activity of CSAct, modification of the native N21 is necessary to prevent loss of ex vivo activity, possibly via protection from degradation.
Sujet(s)
Glycoprotéines/composition chimique , Protéines recombinantes/composition chimique , Aminopeptidases/composition chimique , Aminopeptidases/métabolisme , Animaux , Cerebroside-sulfatase/métabolisme , Chromatographie en phase liquide à haute performance , Clonage moléculaire , Bromure de cyanogène/composition chimique , Disulfures/composition chimique , Fibroblastes/enzymologie , Fibroblastes/métabolisme , Expression des gènes , Glycoprotéines/biosynthèse , Glycoprotéines/déficit , Humains , Cinétique , Masse moléculaire , Structure quaternaire des protéines , Protéines recombinantes/biosynthèse , Diffusion de rayonnements , Spectrométrie de masse ESI , Protéines activatrices des sphingolipides , Sulfoglycosphingolipides/métabolisme , SuidaeRÉSUMÉ
In eubacteria and mitochondria, Hsp70 chaperone activity is controlled by the nucleotide exchange factor GrpE. We have identified the chloroplastic GrpE homolog of Chlamydomonas, CGE1, as an approximately 26-kD protein coimmunoprecipitating with the stromal HSP70B protein. When expressed in Escherichia coli, CGE1 can functionally replace GrpE and interacts physically with DnaK. CGE1 is encoded by a single-copy gene that is induced strongly by heat shock and slightly by light. Alternative splicing generates two isoforms that differ only by two residues in the N-terminal part. The larger form is synthesized preferentially during heat shock, whereas the smaller one dominates at lower temperatures. Fractions of both HSP70B and CGE1 associate with chloroplast membranes in an ATP-sensitive manner. By colorless native PAGE and pulse labeling, CGE1 monomers were found to assemble rapidly into dimers and tetramers. In addition, CGE1 was found to form ATP-sensitive complexes with HSP70B of approximately 230 and approximately 120 kD, the latter increasing dramatically after heat shock.
Sujet(s)
Épissage alternatif , Protéines bactériennes/génétique , Chlamydomonas reinhardtii/génétique , Chloroplastes/métabolisme , Protéines Escherichia coli , Protéines du choc thermique/génétique , Protéines de protozoaire , Séquence d'acides aminés , Animaux , Protéines bactériennes/métabolisme , Séquence nucléotidique , Chlamydomonas reinhardtii/métabolisme , Escherichia coli/génétique , Test de complémentation , Protéines du choc thermique HSP70/génétique , Protéines du choc thermique HSP70/métabolisme , Protéines du choc thermique/métabolisme , Lumière , Données de séquences moléculaires , Mutation , Protéines végétales , Isoformes de protéines , Similitude de séquences d'acides aminésRÉSUMÉ
Purified detergent-soluble cytochrome b6f complex from chloroplast thylakoid membranes (spinach) and cyanobacteria (Mastigocladus laminosus) was highly active, transferring 300-350 electrons per cyt f/s. Visible absorbance spectra showed a red shift of the cytochrome f alpha-band and the Qy chlorophyll a band in the cyanobacterial complex and an absorbance band in the flavin 450-480-nm region of the chloroplast complex. An additional high molecular weight (M(r) approximately 35,000) polypeptide in the chloroplast complex was seen in SDS-polyacrylamide gel electrophoresis at a stoichiometry of approximately 0.9 (cytochrome f)(-1). The extra polypeptide did not stain for heme and was much more accessible to protease than cytochrome f. Electrospray ionization mass spectrometry of CNBr fragments of the 35-kDa polypeptide was diagnostic for ferredoxin:NADP+ oxidoreductase (FNR), as were antibody reactivity to FNR and diaphorase activity. The absence of FNR in the cyanobacterial complex did not impair decyl-plastoquinol-ferricyanide activity. The activity of the FNR in the chloroplast b6f complex was also shown by NADPH reduction, in the presence of added ferredoxin, of 0.8 heme equivalents of the cytochrome b6 subunit. It was inferred that the b6f complex with bound FNR, one equivalent per monomer, provides the membrane protein connection to the main electron transfer chain for ferredoxin-dependent cyclic electron transport.
Sujet(s)
Chloroplastes/enzymologie , Cytochromes de type b/métabolisme , Ferredoxine-NADP reductase/métabolisme , Séquence d'acides aminés , Technique de Western , Clonage moléculaire , Cytochromes de type b/composition chimique , Cytochrome b6f complex , Transport d'électrons , Électrophorèse sur gel de polyacrylamide , Données de séquences moléculaires , Masse moléculaire , Similitude de séquences d'acides aminés , Spectrométrie de masse ESI , Spinacia oleraceaRÉSUMÉ
An accurate, rapid, and versatile method for the analysis of enzyme kinetics using electrospray ionization mass spectrometry (ESI-MS) has been developed and demonstrated using fucosyltransferase V. Reactions performed in primary or secondary amine-containing buffers were diluted in an ESI solvent and directly analyzed without purification of the reaction products. Decreased mass resolution was used to maximize instrument sensitivity, and multiple reaction monitoring (MRM), in the tandem mass spectrometric mode, was used to enhance selectivity of detection. The approach allowed simultaneous monitoring of multiple processes, including substrate consumption, product formation, and the intensity of an internal standard. MRM gave an apparent K(m) for GDP-L-fucose (GDP-Fuc) of 50.4 +/- 5.5 microM and a k(cat) of 1.46 +/- 0.044 s(-1). Under the same conditions, the conventional radioactivity-based assay using GDP-[U-(14)C]Fuc as substrate gave virtually identical results: K(m) = 54.3 +/- 4.6 microM and k(cat) = 1.49 +/- 0.039 s(-1). The close correlation of the data showed that ESI-MS coupled to MRM is a valid approach for the analysis of enzyme kinetics. Consequently, this method represents a valuable alternative to existing analytic methods because of the option of simultaneously monitoring multiple species, the high degree of specificity, and rapid analysis times and because it does not rely on the availability of radioactive or chromogenic substrates.
Sujet(s)
Fucosyltransferases/composition chimique , Osamines/composition chimique , Osamines/métabolisme , Sites de fixation , Substances tampon , Séquence glucidique , Transport d'électrons , Fucose/analogues et dérivés , Fucose/composition chimique , Fucose/métabolisme , Fucosyltransferases/métabolisme , Guanosine diphosphate/analogues et dérivés , Guanosine diphosphate/composition chimique , Guanosine diphosphate/métabolisme , Humains , Concentration en ions d'hydrogène , Ions/composition chimique , Cinétique , Antigènes CD15/analogues et dérivés , Données de séquences moléculaires , Nucléotides/composition chimique , Nucléotides/métabolisme , Spectrométrie de masse ESI/méthodes , Triholosides/composition chimique , Triholosides/métabolismeRÉSUMÉ
Proteins with expanded polyglutamine (polyQ) tracts have been linked to neurodegenerative diseases. One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs). IAs purified from polyQ-expressing cells were dissociated and studied by protein blot assay and mass spectrometry to determine the identity, condition, and relative level of several proteins sequestered within aggregates. Most of the sequestered proteins comigrated with bands from control extracts, indicating that the sequestered proteins were intact and not irreversibly bound to the polyQ polymer. Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle-regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex. These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.
Sujet(s)
Protéines de liaison à l'ADN/métabolisme , Protéines du choc thermique HSP70/métabolisme , Protéines de tissu nerveux/génétique , Protéines nucléaires/génétique , Protéines nucléaires/métabolisme , Peptides/métabolisme , Facteurs de transcription/métabolisme , Ubiquitines/métabolisme , Actines/métabolisme , Sujet âgé , Lignée cellulaire , Taille de la cellule , Corps strié/cytologie , Gènes rapporteurs , Humains , Protéine huntingtine , Méthode TUNEL , Microscopie de fluorescence , Adulte d'âge moyen , Protéines de tissu nerveux/métabolisme , Peptides/génétique , Protéines de fusion recombinantes/métabolisme , Spectrométrie de masse MALDI , Protéine de liaison à la boite TATA , TransfectionRÉSUMÉ
Electrospray ionization mass spectrometry coupled to multiple reaction monitoring (ESI-MS/MRM) has been applied for the first time to analyze enzyme inhibitor kinetics. Specifically, a known competitive inhibitor, guanosine 5'-monophosphate (GMP), and a synthetic, transition-state analogue inhibitor, guanosine 5'-[1D-(1,3,4/2)-5-methyl-5-cyclohexene-1,2,3,4-tetrol 1-diphosphate] (1) have been characterized against recombinant fucosyltransferase (Fuc-T) V using ESI-MS/MRM. Dixon analysis with GMP yielded a signature plot for competitive inhibition. Nonlinear regression analysis gave a Ki of 211.8+/-24.7 microM. The conventional analysis using GDP-[U-14C]-Fuc yielded a similar Ki value of 235.6+/-59.4 microM, confirming the validity of the MS-based method. The synthetic inhibitor 1 showed potent competitive inhibition with a Ki of 25.6+/-2.8 microM. Although 1 possesses a chemically reactive allyl phosphate group, ESI-MS/MRM showed that there was no reduction in the concentration of 1 and no production of a predicted metabolite GDP during the assay. MS/MS also confirmed the absence of a possible pseudo-trisaccharide product. The results clearly show that 1 is neither a slow-reacting donor nor does it act as a suicide-type inhibitor toward Fuc-T V. ESI-MS/MRM is therefore a powerful tool for the kinetic characterization of enzyme inhibitors, providing complete disclosure of the mechanism of action of 1 as an inhibitor.
Sujet(s)
Antienzymes/composition chimique , Guanosine/composition chimique , Fucosyltransferases/antagonistes et inhibiteurs , Fucosyltransferases/composition chimique , Guanosine/analogues et dérivés , Guanosine monophosphate/composition chimique , Indicateurs et réactifs , Cinétique , Analyse de régression , Spectrométrie de masse ESIRÉSUMÉ
As an increasing number of available genomes triggers a gold rush in modern biology, the scientific challenge shifts towards understanding the total of the encoded information, most notably the proteins, their structures, functions and interactions. Currently this work is in its early stages but the near future will bring a merger of biology, engineering and informatics with a far broader impact on society than pure genomics has had so far. The challenge of characterizing the structures and functions of all proteins in a given cell demands technological advances beyond the classical methodologies of protein biochemistry. Mass spectrometry techniques for high-throughput protein identification, including peptide mass fingerprinting, sequence tagging and mass spectrometry on full-length proteins are providing the driving force behind proteomics endeavors. New technologies are needed to move high-resolution protein structure determination to an industrial scale. Nonetheless, improvements in techniques for the separation of intrinsic membrane proteins are enabling proteomics efforts towards identifying drug targets within this important class of biomolecules. Beyond the acquisition of data on sequences, structures and interactions, however, the major work in drug discovery remains: the screening of large candidate compound libraries combined with clever medicinal chemistry that guarantees selective action and defined delivery of the drug.
Sujet(s)
Conception de médicament , Génome humain , Protéome , Électrophorèse bidimensionnelle sur gel , Humains , Spectrométrie de masse , Conformation des protéinesRÉSUMÉ
The cerebroside-sulfate activator protein (CSAct or Saposin B) is a small water-soluble glycoprotein that plays an essential role in the metabolism of certain glycosphingolipids, especially sulfatide. Deficiency of CSAct in humans leads to sulfatide accumulation and neurodegenerative disease. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide-sulfate hydrolysis by arylsulfatase A. CSAct has seven methionine residues and a mass of 8,845 Da when deglycosylated. Mildly oxidized, deglycosylated CSAct (+16 Da), separated from nonoxidized CSAct by reversed-phase high-performance liquid chromatography (RP-HPLC), showed significant modulation of the in vitro activity. Because oxidation partially protected against CNBr cleavage and could largely be reversed by treatment with dithiothreitol, it was concluded that the major modification was conversion of a single methionine to its sulfoxide. High-resolution RP-HPLC separated mildly oxidized CSAct into seven or more different components with shorter retention times than nonoxidized CSAct. Mass spectrometry showed these components to have identical mass (+16 Da). The shorter retention times are consistent with increased polarity accompanying oxidation of surface-exposed methionyl side chains, in general accordance with the existing molecular model. A mass-spectrometric CNBr mapping protocol allowed identification of five of the seven possible methionine-sulfoxide CSAct oxoforms. The most dramatic suppression of activity occurred upon oxidation of Met61 (26% of control) with other residues in the Q60MMMHMQ66 motif falling in the 30-50% activity range. Under conditions of oxidative stress, accumulation of minimally oxidized CSAct protein in vivo could perturb metabolism of sulfatide and other glycosphingolipids. This, in turn, could contribute to the onset and progression of neurodegenerative disease, especially in situations where the catabolism of these materials is marginal.
Sujet(s)
Glycoprotéines/métabolisme , Méthionine/métabolisme , Séquence d'acides aminés , Animaux , Chromatographie en phase liquide à haute performance , Humains , Spectrométrie de masse , Données de séquences moléculaires , Oxydoréduction , Structure tertiaire des protéines , Saposines , Similitude de séquences d'acides aminés , Protéines activatrices des sphingolipidesRÉSUMÉ
The Na(+)/galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni(2+) affinity chromatography and gel filtration. Resequencing the vSGLT gene identified an important correction: the N terminus constitutes an additional 13 functionally essential residues. The mass of His-tagged vSGLT expressed under its native promoter, as determined by electrospray ionization-mass spectrometry (ESI-MS), verifies these 13 residues in wild-type vSGLT. A fusion protein of vSGLT and green fluorescent protein, comprising a mass of over 90 kDa, was also successfully analyzed by ESI-MS. Reconstitution of purified vSGLT yields proteoliposomes active in Na(+)-dependent galactose uptake, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 stoichiometry and apparent K(m) values of 129 mm (Na(+)) and 158 microm (galactose). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monomer. We conclude that vSGLT is a suitable model for the study of sugar cotransporter mechanisms and structure, with potential applicability to the larger SGLT family of important sodium:solute cotransporters. It is further demonstrated that ESI-MS is a powerful tool for the study of proteomics of membrane transporters.
Sujet(s)
Glycoprotéines membranaires/composition chimique , Glycoprotéines membranaires/génétique , Transporteurs de monosaccharides/composition chimique , Transporteurs de monosaccharides/génétique , Vibrio parahaemolyticus/composition chimique , Séquence nucléotidique , Transport biologique , Électrophorèse sur gel de polyacrylamide , Cryofracture , Galactose/pharmacocinétique , Protéines à fluorescence verte , Histidine/métabolisme , Cinétique , Protéines luminescentes/métabolisme , Spectrométrie de masse , Glycoprotéines membranaires/isolement et purification , Microscopie électronique , Données de séquences moléculaires , Transporteurs de monosaccharides/isolement et purification , Plasmides/métabolisme , Régions promotrices (génétique) , Protéolipides/métabolisme , Protéolipides/ultrastructure , Protéines de fusion recombinantes/métabolisme , Sodium/métabolisme , Transporteur-1 sodium-glucose , Facteurs tempsRÉSUMÉ
Cerebroside sulfate activator (CSAct) protein is exceptionally resistant to heat denaturation and proteolytic digestion. Although water soluble the protein binds membrane-associated lipids. Its biological role is thought to be to transfer certain lipids between membranes and to facilitate their catabolism in the lysosomes. An example of the latter is the removal of the sulfate group from cerebroside sulfate by arylsulfatase A. The mechanism of lipid sequestration from membranes and presentation of the lipid-protein complex to catabolic enzymes is a crucial aspect of the function of this protein. The widespread occurrence of the protein class of which CSAct is one of the best known members underscores the significance of this protein. The preparation, purification and chemical and biological properties of a stable disulfide blocked derivative of CSAct is described. The pyridoethylated protein was susceptible to tryptic attack and devoid of a significant population of solvent-protected exchange resistant protons. It apparantly formed a CS complex. However, unlike the complex with the native protein, this was not sufficiently stable to remain intact during size exclusion chromatography. The disulfide-blocked protein had a similar CD spectrum as native protein, indicating similar alpha-helical content. Unexpectedly, the activities of disulfide-blocked protein in the arylsulfatse A catalyzed sulfate hydrolysis from cerebroside sulfate were substantial. Hitherto, it had been assumed that the disulfide connectivities were essential for the protein to maintain a correctly folded configuration to bind lipid ligands and potentiate their hydrolysis. Some revision of our thoughts on the importance of the disulfide connectivities in the structure and function of the protein are necessary.
Sujet(s)
Cérébrosides/métabolisme , Disulfures/composition chimique , Disulfures/métabolisme , Glycoprotéines/composition chimique , Glycoprotéines/métabolisme , Trypsine/métabolisme , Séquence d'acides aminés , Animaux , Cerebroside-sulfatase/métabolisme , Chromatographie sur gel , Chromatographie en phase liquide à haute performance , Dichroïsme circulaire , Glycoprotéines/isolement et purification , Hydrolyse , Cinétique , Ligands , Spectrométrie de masse , Données de séquences moléculaires , Fragments peptidiques/composition chimique , Fragments peptidiques/métabolisme , Liaison aux protéines , Structure secondaire des protéines , Saposines , Protéines activatrices des sphingolipides , Sulfates/métabolisme , Suidae , ThermodynamiqueRÉSUMÉ
Hydrogen-deuterium exchange can be a sensitive indicator of protein structural integrity. Comparisons were made between cerebroside sulfate activator protein (CSAct) in the native state and after treatment with guanidine hydrochloride plus dithiothreitol. Native protein has three internal disulfide bonds and treated protein has no internal disulfide bonds. The comparisons were made using hydrogen-deuterium exchange measured by electrospray ionization mass spectrometry, percentage alpha-helical content measured by circular dichroism and biological activity measured by the ability to support arylsulfatase A-catalyzed sulfate hydrolysis from cerebroside sulfate. In acidic solvent native protein has 59 exchange refractory protons and treated protein has 20 exchange refractory protons (44 and 14% of the exchangeable proton populations, respectively). In native protein the size of the exchange refractory proton population is sensitive to changes in pH, temperature and the presence of a ligand. It is uninfluenced by the presence or absence of glycosyl groups attached to Asn21. Helical content is virtually identical in native and treated protein. Biological activity is significantly reduced but not obliterated in treated protein. The hydrogen-deuterium exchange profile appears to be a sensitive signature of the correctly folded protein, and reflects a dimension of the protein structure that is not apparent in circular dichroic spectra or in the ability of the protein to support arylsulfatase A-catalyzed sulfate hydrolysis from sulfatide. The hydrogen-deuterium exchange profile will be a valuable criterion for characterizing mutant forms of CSAct produced by recombinant and synthetic paradigms and also the native and mutant forms of related proteins.
Sujet(s)
Glycoprotéines/composition chimique , Animaux , Dichroïsme circulaire , Deutérium , Glycoprotéines/métabolisme , Hydrogène , Hydrolyse , Techniques in vitro , Rein/composition chimique , Spectrométrie de masse , Conformation des protéines , Dénaturation des protéines , Protons , Saposines , Protéines activatrices des sphingolipides , Sulfoglycosphingolipides/métabolisme , SuidaeRÉSUMÉ
A general technique has been developed that allows rapid mass spectrometric analysis of full-length membrane proteins [Whitelegge, J. P., le Coutre, J., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10695-10698]. Using in-line HPLC electrospray ionization mass spectrometry (LC-MS), different native and recombinant bacterial membrane proteins of up to 61 kDa are characterized. Mass spectrometric data of four entirely different membrane proteins from three bacterial organisms, two transporters, a channel, and a porin protein are presented. In addition to determination of the molecular mass with an accuracy of +/-0.01%, the technique monitors alkylation or oxidation of single Cys residues and errors in deduced amino acid sequences. Finally, using in-line LC-MS, unknown proteins can be identified from solubilized Escherichia coli membranes without prior purification.
Sujet(s)
Protéines bactériennes/analyse , Protéines bactériennes/composition chimique , Protéines Escherichia coli , Protéines membranaires/analyse , Protéines membranaires/composition chimique , Protéome , Symporteurs , Alkylation , Séquence d'acides aminés , Protéines de la membrane externe bactérienne/analyse , Protéines de la membrane externe bactérienne/composition chimique , Protéines bactériennes/métabolisme , Membrane cellulaire/composition chimique , Chromatographie en phase liquide à haute performance , Cystéine/métabolisme , Escherichia coli/composition chimique , Escherichia coli/cytologie , Ligands , Spectrométrie de masse , Protéines membranaires/métabolisme , Protéines de transport membranaire/analyse , Protéines de transport membranaire/composition chimique , Protéines de transport membranaire/métabolisme , Masse moléculaire , Transporteurs de monosaccharides/analyse , Transporteurs de monosaccharides/composition chimique , Mutation/génétique , Canaux potassiques/analyse , Canaux potassiques/composition chimique , Canaux potassiques/génétique , Canaux potassiques/métabolisme , Protéines de fusion recombinantes/analyse , Protéines de fusion recombinantes/composition chimique , Protéines de fusion recombinantes/métabolisme , Sensibilité et spécificité , Protéines de transport glucose-sodium , Spectrométrie de fluorescence , Marqueurs de spin , Streptomyces/composition chimique , Vibrio parahaemolyticus/composition chimiqueRÉSUMÉ
The specific sugar residues and their linkages in the oligosaccharides from pig kidney and human urine cerebroside sulfate activator proteins (saposin B), although previously hypothesized, have been unambiguously characterized. Exhaustive sequential exoglycosidase digestion of the trimethyl-p-aminophenyl derivatives, followed by either matrix-assisted laser desorption/ionization and/or mass spectrometry, was used to define the residues and their linkages. The oligosaccharides were enzymatically released from the proteins by treatment with peptidyl-N-glycosidase F and separated from the proteins by reversed-phase high-performance liquid chromatography (HPLC). Reducing termini were converted to the trimethyl-p-aminophenyl derivative and the samples were further purified by normal-phase HPLC. The derivatized carbohydrates were then treated sequentially with a series of exoglycosidases of defined specificity, and the products of each digestion were examined by mass spectrometry. The pentasaccharides from pig kidney and human urine protein were shown to be of the asparagine-linked complex type composed of mannose-alpha 1-6-mannose-beta 1-4-N-acetylglucosamine-N-acetylglucosamine(alpha 1-6-fucose). This highly degraded structure probably represents the final product of intra-lysosomal exoglycosidase digestion. Oligosaccharide sequencing by specific exoglycosidase degradation coupled with mass spectrometry is more rapid than conventional oligosaccharide sequencing. The procedures developed will be useful for sequencing other oligosaccharides including those from other members of the lipid-binding protein class to which cerebroside sulfate activator belongs. (c) 2000 John Wiley & Sons, Ltd.
Sujet(s)
Asparagine/composition chimique , Conformation des glucides , Glycoprotéines/composition chimique , Rein/composition chimique , Animaux , Chromatographie en phase liquide à haute performance , Glycoprotéines/urine , Humains , Structure moléculaire , Saposines , Spectrométrie de masse ESI , Spectrométrie de masse MALDI , Protéines activatrices des sphingolipides , SuidaeRÉSUMÉ
Cerebroside sulfate activator protein is a small, heat-stable protein that is exceptionally resistant to proteolytic attack. This protein is essential for the catabolism of cerebroside sulfate and several other glycosphingolipids. Protein purified from pig kidney and human urine was extensively characterized by reversed-phase liquid chromatography and electrospray mass spectrometry. These two sources revealed 20 and 18 different molecular isoforms of the protein, respectively. Plausible explanations of the structures of the majority of these isoforms can be made on the basis of accurate molecular mass assignments. The reversed-phase chromatographic and electrospray mass spectrometric properties of enzymatically deglycosylated and disulfide-reduced protein were also compared. In addition to a demonstration of the power of electrospray ionization mass spectrometry for revealing a wealth of information on protein microheterogeneity and structural detail, the results also demonstrate the utility of this technique for monitoring spontaneous chemical and enzymatically mediated changes that occur as a result of metabolic processing and protein purification.
Sujet(s)
Activateurs d'enzymes/composition chimique , Glycoprotéines/composition chimique , Animaux , Chimie du cerveau , Bovins , Chromatographie en phase liquide à haute performance , Spectroscopie de résonance de spin électronique , Activateurs d'enzymes/isolement et purification , Activateurs d'enzymes/urine , Glucose/composition chimique , Glycoprotéines/isolement et purification , Glycoprotéines/urine , Humains , Rein/composition chimique , Spectrométrie de masse , Oxydoréduction , Saposines , Protéines activatrices des sphingolipides , SuidaeRÉSUMÉ
Genes encoding membrane proteins comprise a substantial proportion of genomes sequenced to date, but ability to perform structural studies on this portion of the proteome is limited. Electrospray ionization-MS (ESI-MS) of an intact protein generates a profile defining the native covalent state of the gene product and its heterogeneity. Here we apply ESI-MS technology with accuracy exceeding 0.01% to a hydrophobic membrane protein with 12-transmembrane alpha-helices, the full-length lactose permease from Escherichia coli. Furthermore, ESI-MS is used to titrate reactive thiols with N-ethylmaleimide. Treatment of the native protein solubilized in detergent micelles reveals only two reactive thiols, and both are protected by a substrate analog.
Sujet(s)
Protéines Escherichia coli , Spectrométrie de masse/méthodes , Protéines membranaires/composition chimique , Protéines de transport membranaire/composition chimique , Transporteurs de monosaccharides , Symporteurs , Chromatographie sur gel/méthodes , Chromatographie en phase liquide à haute performance , Escherichia coli/composition chimique , Facteurs tempsRÉSUMÉ
Purified native Tromp1 was subjected to mass spectrometric analysis in order to determine conclusively whether this protein possesses a cleaved or uncleaved signal peptide. The molecular masses of Tromp1, three Treponema pallidum lipoproteins, and a bovine serum albumin (BSA) control were determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The molecular masses of all of the T. pallidum lipoproteins and BSA were within 0.7% of their respective calculated masses. The molecular mass of Tromp1 was 31,510 Da, which is consistent with a signal-less form of Tromp1, given a calculated mass of unprocessed Tromp1 of 33, 571 Da, a difference of 2,061 Da (a 6.5% difference). Purified native Tromp1 was also subjected to MALDI-TOF analysis in comparison to recombinant Tromp1 following cyanogen bromide cleavage, which further confirmed the identity of Tromp1 and showed that native Tromp1 was not degraded at the carboxy terminus. These studies confirm that Tromp1 is processed and does not contain an uncleaved signal peptide as previously reported.
Sujet(s)
Protéines de la membrane externe bactérienne/analyse , Porines/analyse , Signaux de triage des protéines/analyse , Treponema pallidum/composition chimique , Protéines de la membrane externe bactérienne/génétique , Protéines de la membrane externe bactérienne/métabolisme , Protéines bactériennes , Séquence nucléotidique , Amorces ADN , Porines/génétique , Porines/métabolisme , Signaux de triage des protéines/métabolisme , Protéines recombinantes/analyse , Spectrométrie de masse MALDI , Treponema pallidum/génétique , Treponema pallidum/métabolismeRÉSUMÉ
Cysteine string proteins (csps) are highly conserved constituents of vertebrate and invertebrate secretory organelles. Biochemical and immunoprecipitation experiments implied that vertebrate csps were integral membrane proteins that were tethered to the outer leaflet of secretory vesicles via the fatty acyl residues of their extensively acylated cysteine string. Independently, work of others suggested that Drosophila csps were peripheral membrane proteins that were anchored to membranes by a mechanism that was independent of the cysteine string and its fatty acyl residues. We extended these investigation and found first that sodium carbonate treatment partially stripped both csps and the integral membrane protein, synaptotagmin, from Drosophila membranes. Concomitantly, carbonate released fatty acids into the medium, arguing that it has a mild, solubilizing effect on these membranes. Second, we observed that Drosophila csps behaved like integral membrane proteins in Triton X-114 partitioning experiments. Third, we found that when membrane-bound csps were deacylated, they remained membrane bound. Moreover, it appeared that hydrophobic interactions were necessary for this persistent membrane association of csps. Thus, neither reducing conditions, urea, nor chaotropic agents displaced deacylated csps from membranes. Only detergents were effective in solubilizing deacylated csps. Finally, by virtue of the inaccessibility of deacylated csps to thiol alkylation by the membrane-impermeant alkylating reagent, iodoacetic acid, we inferred that it was the cysteine string domain that mediated the membrane association of deacylated csps. Thus, we conclude that under physiological conditions csps are integral membrane proteins of secretory organelles, and that the cysteine string domain plays a vital role in the membrane association of these proteins.
Sujet(s)
Protéines de liaison au calcium , Drosophila melanogaster , Protéines d'insecte/métabolisme , Protéines membranaires/métabolisme , Protéines de tissu nerveux/composition chimique , Protéines de tissu nerveux/métabolisme , Acylation , Animaux , Carbonates/composition chimique , Membrane cellulaire/composition chimique , Membrane cellulaire/métabolisme , Cyclohexylamines/composition chimique , Dithiothréitol/composition chimique , Protéines du choc thermique HSP40 , Hydroxylamine/composition chimique , Protéines d'insecte/composition chimique , Acide iodo-acétique/composition chimique , Glycoprotéines membranaires/composition chimique , Glycoprotéines membranaires/métabolisme , Protéines membranaires/composition chimique , Octoxinol , Polyéthylène glycols/composition chimique , Acides sulfoniques/composition chimique , SynaptotagminesRÉSUMÉ
Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray-ionization mass spectrometry (ESI-MS) of intact intrinsic membrane proteins purified using reverse-phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the D1 and D2 reaction-center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI-MS study of a G-protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post-translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI-MS experiments.
Sujet(s)
Spectrométrie de masse/méthodes , Protéines membranaires/analyse , Animaux , Apoprotéines/analyse , Bactériorhodopsines/analyse , Bovins , Chloroplastes/ultrastructure , Chromatographie en phase liquide à haute performance , Protéines G/analyse , Halobacterium/composition chimique , Membranes intracellulaires/composition chimique , Complexe protéique du centre réactionnel de la photosynthèse/analyse , Rhodopsine/analyse , Spinacia oleracea/ultrastructureRÉSUMÉ
Photosystem II catalyzes the photooxidation of water to molecular oxygen, providing electrons to the photosynthetic electron transfer chain. The D1 and D2 chloroplast-encoded reaction center polypeptides bind cofactors essential for Photosystem II function. Transformation of the chloroplast genome of the eukaryotic green alga Chlamydomonas reinhardtii has allowed us to engineer site-directed mutants in which aspartate residue 170 of D1 is replaced by histidine (D170H), asparagine (D170N), threonine (D170T), or proline (D170P). Mutants D170T and D170P are completely deficient in oxygen evolution, but retain normal (D170T) or 50% (D170P) levels of Photosystem II reaction centers. D170H and D170N accumulate wild-type levels of PSII centers, yet evolve oxygen at rates approximately 45% and 15% those of control cells, respectively. Kinetic analysis of chlorophyll fluorescence in the mutants reveals a specific defect in electron donation to the reaction center. Measurements of oxygen flash yields in D170H show, however, that those reaction centers capable of evolving oxygen function normally. We conclude that aspartate residue 170 of the D1 polypeptide plays a critical role in the initial binding of manganese as the functional chloroplast oxygen-evolving complex is assembled.