Different conformational changes within the F-helix occur during serpin folding, polymerization, and proteinase inhibition.

The intrinsic metastability of the serpin native state is the thermodynamic driving force for both proteinase inhibition and the formation of inactive polymers. A number of mechanisms has been proposed to explain how both these conformational changes are achieved. However, one aspect that has received little attention is the movement of the F-helix, which physically impedes both these events. We have applied a protein engineering approach to investigate the conformational changes of this helix during proteinase inhibition, serpin folding, and polymerization. We systematically mutated two highly conserved hydrophobic residues on the F-helix, V161 and I157, and in addition, removed a hydrogen bond between D149 and the first turn of the helix. Our data demonstrate that while all three interactions are important for the stability and folding of the molecule, their contribution during inhibition and polymerization differ. The presence of I157 is crucial to all conformational changes as its loss results in inactivation of the serpin and rapid polymerization. The replacement of D149 does not affect activity but significantly increases the polymerization rate. The interactions formed by V161 play an important role only in maintaining the native conformation. Taken together, these data suggest that the F-helix undergoes a reversible conformational change in both its N- and C-termini during proteinase inhibition only the C-terminus undergoes changes during polymerization, but there is a global change required for folding.