Search PubMed⌕ Search

Biomedical subjects

G Padron

Publications and source records attributed to G Padron.

13 recordsLinked to original sources
PubMed2000

Automated interpretation of low-energy collision-induced dissociation spectra by SeqMS, a software aid for de novo sequencing by tandem mass spectrometry.

SeqMS, a software aid for de novo sequencing by tandem mass spectrometry (MS/MS), which was initially developed for the automated interpretation of high-energy collision-induced dissociation (CID) MS/MS spectra of peptides, has been applied to the interpretation of low-energy CID and post-source decay (PSD) spectra of peptides. Based on peptide backbone fragmented ions and their related ions, which are the dominant ions observed in the latter two techniques, the types of ions and their propensities to be observed have been optimized for efficient interpretation of the spectra. In a typical example, the modified SeqMS allowed the complete sequencing of a 31-amino acid synthetic peptide, except for the isobaric amino acids (Leu or Ile, and Lys or Gln), based on only the low-energy CID-MS/MS spectrum.

Amino Acid Sequence↗
PubMed1999

Structural characterization of Acetobacter diazotropicus levansucrase by matrix-assisted laser desorption/ionization mass spectrometry: identification of an N-terminal blocking group and a free-thiol cysteine residue.

High-resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize the primary structure of the levansucrase (EC 2.4.1.10) secreted by Acetobacter diazotropicus SRT4. The technique permitted not only the reading frame of this enzyme, the amino acid sequence of which was deduced from DNA, but also the elucidation of an N-terminal blocking group and the position of a disulfide bridge between Cys309 and Cys365 among the three Cys residues. A free cysteine (Cys127) was identified by modifying an intact molecule with a sulfhydryl reagent, 5-(octyldithio)-2-nitrobenzoic acid, under non-reducing conditions. In addition, the enzyme obtained by site-directed mutagenesis at Asp279 to Asn279 was also identified by the above methods. Post-source decay analysis of the tryptic peptide containing the mutation site unequivocally revealed an Asn residue at position 279.

Acetobacter↗
PubMed1998

Automated interpretation of high-energy collision-induced dissociation spectra of singly protonated peptides by 'SeqMS', a software aid for de novo sequencing by tandem mass spectrometry.

SeqMS, a software program designed for the automated interpretation of high-energy collision-induced dissociation (CID) mass spectra of singly protonated peptides ionized by fast atom bombardment, has been developed. The software is capable of probing the sequence of an unknown peptide, and even of certain modified peptides. The program, compiled for WINDOWS95 or NT, also permits the retrieval of raw data and the reconstruction of the spectra on a user-friendly graphical interface with the aid of several tools for processing the spectra, which include setting multiple threshold levels and automatic peak detection. SeqMS is capable of generating candidate sequences, based on the detected peaks, and of displaying the resulting assignments for each candidate in a spectrum or in tabular form. The software has the following capabilities: 1) the ions derived from backbone and side-chain fragmentations, internal and immonium ions, and side-chain loss ions can be used for calculation; 2) 18O-labeling of a peptide at the C terminus, a methodology which was developed to differentiate N-terminal from C-terminal ions, is applicable as an optional setting; 3) modified amino acids and N- or C-terminal blocking groups are taken into account for calculation according to the user's setting in a library; 4) amino acid composition and partial or complete amino acid sequence of a peptide can be used as input for calculation; 5) the assignments of signal output in a spectrum can be graphically edited, and then re-calculated based on the edited peaks. The efficacy of the program is demonstrated by testing 74 high-energy CID spectra, obtained using a four-sector instrument, of synthetic, proteolytic, and biologically active peptides, some of which contain modified groups.

Algorithms↗
PubMed1996

Effect of the position of a basic amino acid on C-terminal rearrangement of protonated peptides upon collision-induced dissociation.

Internal rearrangement involving the loss of the C-terminal amino acid residue upon collision-induced dissociation (CID) or metastable decomposition was studied for protonated peptides. To investigate the structural characteristics of peptides responsible for this rearrangement, a series of synthetic peptides were prepared and subjected to B/E-linked scan or tandem mass spectrometric analyses using a four-sector instrument. The results showed that the position of a basic amino acid in the peptide sequence and its basicity have a significant influence on the rearrangement. Arginine (Arg) located at the n-1 position facilitates the rearrangement with about twice as many rearrangement ions as is observed for the other Arg-containing peptides. This can be attributed to the interaction of a positively charged guanidino group of Arg with its own carbonyl group via a salt bridge which is tightly formed in vacuo between a guanidino and carboxylate groups, the mechanism of which is analogous to that previously proposed for the formation of similar rearrangement ions observed in the spectra of metal-cationized peptides. This association would result in the facile attack of the C-terminal hydroxyl group on the penultimate carbonyl group, leading to the rearrangement. In addition, the rearrangement ion was observed both in metastable decomposition and high-energy CID spectra obtained by B/E-linked scan analyses without or with gas, respectively, but in a sequence dependent manner.

Amino Acid Sequence↗
PubMed1995

The use of position-specific rotamers in model building by homology.

In this study we concentrate on replacing side chains as a subtask of model building by homology. Two problems arise. How to determine potential low energy rotamers? And how to avoid the combinatorial explosion that results from the combination of many residues for which multiple good rotamers are predicted? We attempt to solve these problems by choosing position-specific rather than generalized rotamers and by sorting the residues that have to be modelled as a function of their freedom in rotamer space. The practical advantages of our method are the quality of the models for cases of high backbone similarity, the small amount of human intervention needed, and the fact that the method automatically estimates the reliability with which each residue has been modeled. Other methods described in this issue are probably more suitable if large backbone rearrangements or loop insertions and deletions need to be modeled.

Bacterial Proteins↗
PubMed1993

Protein flexibility and aggregation state of human epidermal growth factor. A time-resolved fluorescence study of the native protein and engineered single-tryptophan mutants.

A time-resolved fluorescence spectroscopic study of the recombinant human epidermal growth factor (hEGF), a bis(tryptophan)-containing protein (Trp49-Trp50), and of the two single-tryptophan-containing engineered mutants with Trp49 or Trp50 replaced by Phe ([W49F]hEGF, [W50F]hEGF), was undertaken in order to gain insight into the conformational dynamics of the C-terminal region. Quite different position-dependent microenvironments for the two Trp residues are shown by comparing the fluorescence intensity decay of both mutants. Trp50 in the single-tryptophan mutant [W49F]EGF probably undergoes a dominant interaction with the solvent. A more heterogeneous environment of Trp49 in the [W50F]hEGF mutant is found. Moreover, the fluorescence decay of the native hEGF is not simply the additive result of the decays of both mutants: the Trp2 sequence confers a conformation of the C-terminal sequence which is more in contact with the rest of the protein molecule. By contrast, the fluorescence anisotropy decay of the native protein is quite similar to that of the single-tryptophan mutants. A high degree of rotational freedom in the C-terminal region of the protein is demonstrated. The resonance energy transfer, which could contribute to the anisotropy decay, appears therefore not to be highly efficient with respect to the depolarization motions. In addition to these local conformational and dynamic aspects of the hEGF C-terminal sequence, the fluorescence anisotropy decay data demonstrate the existence of a dimerization process of the native protein which is dependent on pH and protein concentration. This phenomenon influences the excited-state lifetime profiles and, therefore, the local conformational equilibrium of the C-terminal region.

Epidermal Growth Factor↗
PubMed1992

Time-resolved fluorescence study of human recombinant interferon alpha 2. Association state of the protein, spatial proximity of the two tryptophan residues.

Human recombinant interferon alpha 2 belongs a to family of proteins active against a wide range of viruses. It contains two tryptophan residues located at positions 77 and 141 in the peptide sequence. The fluorescence emission spectrum of these tryptophan residues displays a maximum at 335 nm. The fluorescence intensity decay is described by one broad excited-state-lifetime population centered around a value of 1.7 ns (full width at half maximum, 1.5 ns). These observations suggest that in the native protein, both tryptophan residues emit from similar environments, not directly exposed to the surrounding solvent. The anisotropy decay is essentially biexponential. The correlation-time value characterizing the Brownian rotation of the protein varies linearly with the viscosity/temperature ratio. The calculated hydrodynamic volumes are compatible with the existence of a dimer and a tetramer, at pH 5.5 and 9.4, respectively. Addition of urea at pH 5.5 disrupts the dimer and modifies to some extent the excited-state-lifetime distribution which becomes more heterogeneous. Disulfide-bond reduction also dissociates the dimer and leads to a highly heterogeneous fluorescence-intensity decay with four excited-state-lifetime populations. An opening of the local structure in the Trp region of the protein is likely to occur in these conditions. The fast-anisotropy-decay components can be due to either fast rotation or energy transfer between the indoles. Close proximity of the two Trp residues (less than 1 nm) is suggested from steady-state and time-resolved fluorescence-anisotropy measurements in vitrified medium [95% (by mass) glycerol at -38 degrees C]. This suggestion is in agreement with the recently published three-dimensional structure of the homologous protein murine interferon beta [Senda, T., Shimazu, T., Matsuda, S. Kawano, G., Shimizu, H., Nakamura, K. T. & Mitsui, Y. (1992) EMBO J. 11, 3193-3201].

Fluorescence Polarization↗
PubMed1992

A method for determination of N-glycosylation sites in glycoproteins by collision-induced dissociation analysis in fast atom bombardment mass spectrometry: identification of the positions of carbohydrate-linked asparagine in recombinant alpha-amylase by treatment with peptide-N-glycosidase F in 18O-labeled water.

Previously, a combined use of fast atom bombardment (FAB) mass spectrometry and peptide N-glycosidase F, an enzyme that cleaves the beta-aspartylglycosylamine linkage of Asn-linked carbohydrates, was successfully applied to identification of N-glycosylation sites in a glycoprotein with the known or DNA-derived sequence (S. A. Carr and G. D. Roberts, 1986, Anal. Biochem. 157, 396-406). Here, we extended the method for easier identification of N-glycosylation sites in a glycoprotein even with unknown sequence. The glycoprotein is digested with peptide-N-glycosidase F in buffer containing 40 at% H2 18O, to yield a deglycosylated protein whose carbohydrate-linked Asn residues are converted to Asp partly labeled with 18O at their beta-carboxyl group during this digestion. The deglycosylated protein is further digested with proteolytic enzymes in an appropriate buffer prepared with normal water, and then peptides are separated on a reversed-phase column by HPLC. Peptides in which carbohydrate-linked Asn has been converted to Asp show a pair of signals ([M + 1]+ and [M + 3]+) in FAB mass spectra due to the partial incorporation of 18O into the beta-carboxyl groups of Asp residues, while the other peptides show normal isotopic ion distributions. Thus, both formally N-glycosylated peptides and, using collision-induced dissociation analysis, N-glycosylation sites can be identified. The application of the present method to the determination of N-glycosylation sites in a recombinant glycoprotein, Bacillus licheniformis alpha-amylase, is described.

Amidohydrolases↗
PubMed1992

Interaction of recombinant human epidermal growth factor with phospholipid vesicles. A steady-state and time-resolved fluorescence study of the bis-tryptophan sequence (Trp49-Trp50).

The interaction of recombinant human epidermal growth factor with small unilamellar phospholipid vesicles was studied by steady-state and time-resolved fluorescence of the bis-tryptophan sequence (Trp49-Trp50). Steady-state anisotropy measurements demonstrate that strong binding occurred with small unilamellar vesicles made up of acidic phospholipids at acidic pH only (pH < or = 4.7). An apparent stoichiometry for 1,2-dimyristoyl-sn-phosphoglycerol of about 12 phospholipid molecules per molecule of human epidermal growth factor was estimated. The binding appears to be more efficient at temperatures above the gel to liquid-crystalline phase transition. The conformation and the environment of the Trp-Trp sequence are not greatly modified after binding, as judged from the invariance of the excited state lifetime distribution and from that of the fast processes affecting the anisotropy decay. This suggests that the Trp-Trp sequence is not embedded within the bilayer, in contrast to the situation in surfactant micelles (Mayo et al. 1987; Kohda and Inigaki 1992).

Amino Acid Sequence↗
PubMed1990

Production and characterization of human gamma interferon from Escherichia coli.

The production of human gamma interferon as intracellular inclusion bodies in Escherichia coli, which simplified the purification process, is described. An expression plasmid carrying lipoprotein and the tryptophane promoters in tandem was used. Preparation of highly pure interferon was achieved using high resolution chromatography after denaturation and renaturation steps. Structural characteristics of this protein were verified by mass spectrometric analysis. Additional control tests have shown the suitability of the final product for clinical purposes.

Amino Acid Sequence↗
PubMed1989

Biologicals production by recombinant DNA technology in Cuba.

During 1981 we started an intensive programme for the development of biotechnology in Cuba. The first project was related to the production of leucocyte interferon and the cloning an expression of these genes in prokaryotic and yeast cells. These products are currently obtained in large amounts and their physical and chemical characterization are presented. The use of mammalian cells in culture for the production of recombinant proteins has also been carried out. The results obtained with the expression and amplification of the hepatitis B surface antigen (HBsAg) in CHO cells are discussed and compared with that obtained in yeast. The construction of a recombinant vaccinia virus carrying the HBsAg was also achieved. The specific transcripts were characterized and the Ab levels tested in animals. The results presented here indicate that the host system is a very important aspect to be taken into consideration and in some cases mammalian cells appear to be the best choice.

Animals↗