Local unfolding is required for the site-specific protein modification by transglutaminase.
The transglutaminase (TGase) from Streptomyces mobaraensis catalyzes transamidation reactions in a protein substrate leading to the modification of the side chains of Gln and Lys residues according to the A-CONH(2) + H(2)N-B → A-CONH-B + NH(3) reaction, where both A and B can be a protein or a ligand. A noteworthy property of TGase is its susbstrate specificity, so that often only a few specific Gln or Lys residues can be modified in a globular protein. The molecular features of a globular protein dictating the site-specific reactions mediated by TGase are yet poorly understood. Here, we have analyzed the reactivity toward TGase of apomyoglobin (apoMb), α-lactalbumin (α-LA), and fragment 205-316 of thermolysin. These proteins are models of protein structure and folding that have been studied previously using the limited proteolysis technique to unravel regions of local unfolding in their amino acid sequences. The three proteins were modified by TGase at the level of Gln or Lys residues with dansylcadaverine or carbobenzoxy-l-glutaminylglycine, respectively. Despite these model proteins containing several Gln and Lys residues, the sites of TGase derivatization occur over restricted chain regions of the protein substrates. In particular, the TGase-mediated modifications occur in the "helix F" region in apoMb, in the β-domain in apo-α-LA in its molten globule state, and in the N-terminal region in fragment 205-316 of thermolysin. Interestingly, the sites of limited proteolysis are located in the same chain regions of these proteins, thus providing a clear-cut demonstration that chain flexibility or local unfolding overwhelmingly dictates the site-specific modification by both TGase and a protease.
CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy. barbara.spolaore@unipd.it
Barbara
B
Spolaore
Samanta
S
Raboni
Amparo
A
Ramos Molina
Abhijeet
A
Satwekar
Nunzio
N
Damiano
Angelo
A
Fontana
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology; D002417:Cattle; D006736:Horses; D007768:Lactalbumin / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D058767:Protein Unfolding; D059748:Proteolysis; D013302:Streptomyces / Q000201:enzymology*; D013820:Thermolysin / Q000737:chemistry / Q000378:metabolism*; D011503:Transglutaminases / Q000378:metabolism*
D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin; D007768:Lactalbumin; D011503:Transglutaminases; D013820:Thermolysin