Reference "Probing the modelled structure of wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures"

Reference ID

9219

Title

Probing the modelled structure of wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures

Source

Proteins, 1999, vol. 36, pp. 192-204

Authors (8)

Caporale-C	Caruso-C
Facchiano-A	Nobile-M
Leonardi-L	Bertini-L
Colonna-G	Buonocore-V

Abstract

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 wheatwin1, 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.