Investigation for transition state of loop insertion in serpin : An approach toward prevention of amyloidosis

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A conformational change, loop-insertion of serine proteinase inhibitors (serpin) may cause protein coagulation in certain tissues in the cases of their inherited molecular-abnormalities. In order to control the loop-insertion, a supposed transition state has been investigated in the present project. Ovalbumin, the albumen protein, which belongs to a superfamily of serpin has been focused although it does not show any inhibitory activity to serine proteinases. Using some ovalbumin mutants, improvement in quantitative determination of loop-insertion has been attempted. Previously, loop-insertion of the ovalbumin mutant had been observed by measuring time-courses of limited proteolysis with subtilisin. Since the procedure had been complicated and erroneous, some quantitative and convenient methods have been longed. As the ion-exchanger column chromatography on HPLC can differentiate the conformational isomer through the serpin loop-insertion, we set up a novel procedure for quantitative analysis of loop-insertion in ovalbumin mutants in the present project. Utilizing an ovalbumin mutant R339T/A352R in which the P1-P1' site is accessible against trypsin, the novel HPLC procedure has been proved to be a simple and accurate procedure. Because of the structural and functional situations of serpin, increased loop insertion rate should lead to the acquisition of the inhibitory activity. We therefore did further mutagenesis to accelerate the loop insertion rate on the basis of the data of crystal structure. Additional mutants, K290T/R339T/A352R and R104A/R339T/A352R, and the disulfide-reduced form of R339T/A352R, respectively, displayed 1.5, 3.7, and 6.7-fold increase in the loop insertion rate as compared with R339T/A352R control. The mutation and disulfide reduction should give a more flexible nature on the distal sheet A structure.