The unusual shutter region of ZPI. ZPI has a negatively charged D213 buried in the hydrophobic shutter region, where it forms only 1 hydrogen bond with neighboring Q363 (A). Antitrypsin has a different hydrogen-bonding network around the shutter region. This position is occupied by a conserved Asn186, which forms 4-hydrogen bonds with neighboring residues (B). The labeled residues are shown in sticks with carbon atoms in green, nitrogen in blue, and oxygen in red. Replacement of D213 of ZPI with Asn increased the T_m of ZPI by more than 10°C, as measured by circular dichroism (C), whereas D213A mutant has the same T_m as that of wild-type ZPI.