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Probing the modelled structure of Wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures
Author(s) -
Caporale Carlo,
Caruso Carla,
Facchiano Angelo,
Nobile Monica,
Leonardi Luca,
Bertini Laura,
Colonna Giovanni,
Buonocore Vincenzo
Publication year - 1999
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(19990801)36:2<192::aid-prot5>3.0.co;2-l
Subject(s) - proteolysis , hydrolysis , sequence (biology) , peptide sequence , chemistry , digestion (alchemy) , biochemistry , sequence analysis , peptide , protein superfamily , protein structure , computational biology , amino acid , hydrolyzed protein , proteases , biology , chromatography , enzyme , gene
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.