SERPIN CONFORMATIONAL CHANGE IN OVALBUMIN - ENHANCED REACTIVE CENTER LOOP INSERTION THROUGH HINGE REGION MUTATIONS

Ovalbumin is a noninhibitory member of the serpin superfamily that doe s not spontaneously undergo the loop-to-sheet conformational change up on cleavage of its reactive center that is characteristic of inhibitor y serpins, We tested the hypothesis that ovalbumin could be turned int o a proteinase inhibitor by increasing the rate of loop insertion thro ugh hinge region mutations alone. We found that none of the three vari ants examined showed any detectable proteinase inhibitory properties, However, replacement of the P14 arginine residue of ovalbumin by serin e, either alone or in combination with changes of P12-P10 to alanine, resulted in a large increase in the rate of loop insertion into beta-s heet A following cleavage at the P1-P1' bond by porcine pancreatic ela stase (PPE), as shown by the spontaneous formation of a loop-inserted form upon cleavage that has increased the thermal stability, From the magnitude of the increase in stability of the cleaved, loop-inserted f orms of the P14 ovalbumin variants, as well as the accessibility of th e P1-P1'-cleaved reactive center loop to further proteolysis at P8-P7, we concluded that the reactive center loop can only partially insert into beta-sheet A and therefore that ovalbumin is also defective in th e ability of beta-sheet A to expand to fully accommodate the whole of the reactive center loop. This defect, through its effect on the exten t and/or rate of loop insertion, is likely to be a principal reason fo r ovalbumin not being a proteinase inhibitor.