Local Unfolding Is Required
for the Site-Specific
Protein Modification by Transglutaminase
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Abstract
The transglutaminase (TGase) from <i>Streptomyces
mobaraensis</i> 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<sub>2</sub> + H<sub>2</sub>N-B → A-CONH-B + NH<sub>3</sub> 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