Probing the modelled structure of wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures.
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.
Caporale
C
C
Dipartimento di Agrobiologia ed Agrochimica, Università della Tuscia, Viterbo, Italy. caporale@unitus.it
Caruso
C
C
Facchiano
A
A
Nobile
M
M
Leonardi
L
L
Bertini
L
L
Colonna
G
G
Buonocore
V
V
D000478:Alkylation; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D001467:Hordeum; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D013381:Subtilisins / Q000378:metabolism; D014908:Triticum; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D010940:Plant Proteins; C062880:Win-1 proteins, plant; C076873:barwin protein, Hordeum vulgare; D010450:Endopeptidases; C044499:submandibular proteinase A; D012697:Serine Endopeptidases; D013381:Subtilisins; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin