Analytical techniques and computer algorithms combined for the rapid characterization of structural peptide and protein features

Caporale, Carlo

doi:10.1111/j.1399-3011.1998.tb00666.x

Cited by 4 publications

References 44 publications

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Probing the modelled structure of Wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures

Caporale¹,

Caruso²,

Facchiano³

et al. 1999

Proteins

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We set up a method to get rapid information on the three-dimensional structure of peptide and proteins of known sequence. Both native and alkylated polypeptide is hydrolyzed with a number of proteases at different digestion times and the resulting mixtures are compared by HPLC analysis to establish the differences in the hydrolysis pathways of the folded and unfolded molecule. Then, the unfractionated digestion mixtures of the native polypeptide are submitted to automatic sequence analysis to identify the hydrolysis sites. The sequence of each fragment present in the mixtures is reconstructed and its amount determined by quantitative data of the sequence analyses. We used this approach to determine the amino acid surface accessibility of wheat-win1, a pathogenesis-related protein from wheat, and constructed a predictive three-dimensional model based on the knowledge of the tertiary structure of barwin, a highly homologous protein from barley. The procedure allowed us to quickly identify and quantify the hydrolysis at the susceptible bonds which could be classified as exposed , partially hidden, or inaccessible. The results were useful to evidentiate and discuss concordances and differences between experimental and model predicted accessibilities of amino acid residues. Proteins 1999;36:192-204. 1999 Wiley-Liss, Inc.

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Probing the modelled structure of Wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures

Caporale¹,

Caruso²,

Facchiano³

et al. 1999

Proteins

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STAT: A Saccharide Topology Analysis Tool Used in Combination with Tandem Mass Spectrometry

2000

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Sequential stages of mass spectrometry (MSn) have the potential to provide a great deal of structural information in glycan analysis. The saccharide topology analysis tool (STAT) presented here is a Web-based computational program that can quickly extract sequence information from a set of MSn spectra for an oligosaccharide of up to 10 residues. After information such as precursor ion mass, possible monosaccharide moieties, charge carrier, and product ion mass has been input, all possible connectivities are generated and evaluated against the MSn data. The list of possible structures is given a rating based on the likelihood that it is the correct sequence. Examples are given to demonstrate the feasibility of applying STAT to MSn data generated from bacterial lipooligosaccharides and an N-linked glycan. The major advantage of STAT is that the list of possible structures is generated quickly and the rating system pushes the more likely structures to the top of the list. Combining the data generated by STAT with data on the branching patterns of the glycan serves to eliminate all but a handful of structures. These remaining structures could then be used to guide further structural analysis.

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Isolation and Amino Acid Sequence of Two New PR-4 Proteins from Wheat

et al. 2001

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We have purified and characterized two new pathogenesis-related (PR) proteins from wheat belonging to the PR-4 family. We named the proteins wheatwin3 and wheatwin4 in analogy with the previously characterized wheatwin1 and wheatwin2. Their isoelectric points were 7.1 and 8.4, respectively. We determined the complete amino acid sequence of both proteins by a rapid approach based on the knowledge of the primary structures of the homologous wheatwin1 and wheatwin2. Wheatwin3 differs from wheatwin1 in one substitution at position 88, while wheatwin4 differs from wheatwin2 in one substitution at position 78. The secondary structure and solvent accessibility of these residues were determined on the three-dimensional model of wheatwinl. Residue 88 was very accessible and was located in a flexible region. Preliminary results indicate that, like wheatwin1 and wheatwin2, wheatwin3 and wheatwin4 have antifungal activity.

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Analytical techniques and computer algorithms combined for the rapid characterization of structural peptide and protein features

Cited by 4 publications

References 44 publications

Probing the modelled structure of Wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures

Probing the modelled structure of Wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures

STAT: A Saccharide Topology Analysis Tool Used in Combination with Tandem Mass Spectrometry

Isolation and Amino Acid Sequence of Two New PR-4 Proteins from Wheat

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