Several variants of al-proteinase inhibitor (al-PJ) were investigated by spectroscopic methods and characterized according to their inhibitory activity. Replacement of Thr345 (P14) a,-Proteinase inhibitor (al-PI) is a member of the serine proteinase inhibitor (serpin) family [l -41. Inhibitors of this class play crucial roles in controlling proteolytic activities of major physiological importance, such as in coagulation, fibrinolysis and complement activation [5]. Several studies provided evidence that serpins are recognized by their target proteinases as substrates and that cleavage occurs between their P1 and PI' residues in the active site (positions 358 and 359 in al-PI) leading to a major conformational change in human al-P1 [6 -12al. More detailed information on how this conformational transition might take place was obtained from X-ray crystallographic analysis of the three-dimensional structure of al-PI cleaved at its active site by chymotrypsinogen. The most striking feature of this structure was that residues Met358 (Pl) and Ser359 (Pl') (see Fig. 1B) were separated by approximately 7 nm in the cleaved inhibitor [6]. It was hypothesized that insertion of residues 345 -358 of native a l -P1 into a pre-existing p-sheet in an antiparallel fashion was the key event of this conformational change. More recent studies on the structure of ovalbumin, a serpin with no inhibitory activity, and its cleaved form plakalbumin (see Fig. 1 A), which do not show this conformational change, confirmed this hypothesis and provided experimental evidence for the conformation of an uncleaved serpin [9, 131. In the follow-
MATERIALS AND METHODS
Production of a,-PI variantsOligonucleotide directed mutagenesis of the cDNA coding for [Met358 -+ Arg]al-PI with a deletion of its five N-termind amino acids was performed as previously described [15, 161. Expression of [Thr345 4 Arg, Met358 + ArgJal-Pi, [Met351 --$ Glu, Met358 -+Arg]a,-PI and [Met358 +Arg]al-PI was carried out in Escherichia coli (strain TGE 901) from plasmids pTG 7952, pTG 7955 and pTG 1943, respectively, under the control of the leftward promoter of phage 1 . in the presence of a temperature-sensitive repressor cl. For increased expression, a synthetic ribosome-binding site was employed [17]. Cultures were grown under selective conditions (200 mg/l ampicillin) at 30°C in a 20-1 fermenter (LSL Biolafitte, Saint-Germain en Laye, France) to an A600 of 10. Expression was induced by shifting the growth temperature to 42°C for 6 h. Isolation of the a,-PI variants was performed as previously described [18].