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

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 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.

Wheat pathogenesis‐related proteins of class 4 have ribonuclease activity

“…Furthermore, like the highly homologous protein barwin (95, 2% identity, Fig. 4C), its structure consists of a main beta‐sheet of four anti‐parallel strands, two short parallel beta strands constituting a little independent beta‐sheet, and few short helices [10,11,14]. Consequently, the ribonuclease activity of wheatwin1 can be explained by the classical acid–base mechanism common to other ribonucleases involving two His residues [16, and references therein].…”

“…3D structure models of wheatwin1 were based on the availability of the NMR three‐dimensional co‐ordinates of the homologous protein barwin [10–12] (PDB code 1BW3) and performed as previously described [14,15]. The alignment of wheatwin1 and barwin did not require deletion or insertion of gap.…”

“…We demonstrated that wheatwin1 and wheatwin2 and their genes are specifically induced in wheat seedlings infected with Fusarium culmorum and upon treatment with systemic acquired resistance activators [6,7]; the cDNAs have been cloned and the recombinant proteins expressed in E. coli [8,9]. The three‐dimensional model of wheatwin1 has been already designed (PDB code 1C2Z) and experimentally validated [14], based on the knowledge of the tertiary structure in solution of barwin [10,11, PDB code 1BW3], a highly homologous protein from barley [12] showing a six‐stranded double‐psi ß barrel [13]. We also compared the 3D structures of the four wheatwin proteins by homology modelling and related their micro differences to the different antifungal activity [15].…”

Alteration in the ubiquitin structure and function in the human lens: a possible mechanism of senile cataractogenesis

“…4), whereas the N‐terminus segment is involved in an internal strand of the β‐sheet. The disordered region at the C‐terminus is well suitable for proteolytic cleavage, as demonstrated by different authors [13–15]. In these articles, it was demonstrated that beta strands are not hydrolyzed by proteases (even if this opportunity could not be excluded for the external strands, which may leave the β‐sheet, whereas the internal strands are very strongly constrained).…”

Modeling the 3D structure of wheat subtilisin/chymotrypsin inhibitor (WSCI). Probing the reactive site with two susceptible proteinases by time-course analysis and molecular dynamics simulations

“…BLAST sequence alignment of WSCI and the reference protein was used to build the models, which had sequence identity of 87% without gaps. According to a procedure used in similar previous studies (Caporale et al, 1999;Facchiano et al, 2001;Costantini et al, 2005;Marabotti and Facchiano, 2005) and in agreement with suggestions in a recent review of protein comparative modeling (Wallner and Elofsson, 2005), we created 10 full-atom models of WSCI by setting 4.0 Å as the root mean square deviation (RMSD) among initial models and by full optimization of models, i.e., multiple cycles of refining with conjugate gradient minimization and molecular dynamics with simulated annealing. According to a procedure used in similar previous studies (Caporale et al, 1999;Facchiano et al, 2001;Costantini et al, 2005;Marabotti and Facchiano, 2005) and in agreement with suggestions in a recent review of protein comparative modeling (Wallner and Elofsson, 2005), we created 10 full-atom models of WSCI by setting 4.0 Å as the root mean square deviation (RMSD) among initial models and by full optimization of models, i.e., multiple cycles of refining with conjugate gradient minimization and molecular dynamics with simulated annealing.…”