Product conformation driven splicing of unprotected peptides by reverse proteolysis: influence of intrinsic and extrinsic factors.

The structural motif of 'product conformation driven V8 protease catalyzed ligation reaction' can be represented by FR(I)-EALER-FR(II). The relative roles of the flanking regions (FR(I) and FR(II)) and of splicedon, the central penta-peptide, on the thermodynamic stability of the 'conformational trap' of the product has been now evaluated as a function of co-solvent concentration. The studies have established that the thermodynamic stability of the conformational trap of alpha17-40des23-26 with four different splicedons (EALER, EALEV, EYGER, or EGAER) that differ in the intrinsic alpha-helical potential of their amino acid residues and/or ability to generate i, i+4 side chain interaction is a direct correlate of the n-propanol induced alpha-helical conformation of the product. On the other hand, when the product is defined by only splicedon EALER, and the flanking regions are disitinct; no correlation could be drawn between the stability of the trap and solvent induced alpha-helical conformation, even though these generally give an equilibrium yield of 45% in 30% n-propanol and is not influenced by an increased propanol concentration. However, when the splicedon EALER with given FR(I) and FR(II) region develops a 'conformational trap' of a lower stability in 30% propanol as seen with beta18-25(A22)-EALER-beta31-39, the stability increases in 60% n-propanol, without significant increase in the alpha- helical conformation. Though, primary structure of RNAse1-20, could be presented as RNAse1-5-AKFER- RNAse1-20, and alpha-helical conformation is induced to this peptide both in 30 and 60% propanol, splicedon AKFER by itself does not develop the 'conformational trap' of RNAse1-20. The splicedon AKFER of RNAse1-20 fails to develop the 'conformational trap', due to an intrinsic inhibitory potential of its FR region, RNAse11-20; replacing RNAse11-20 with alpha32-40 enables the splicedon AFKER to generate the 'conformational trap'. The studies presented here have demonstrated the primary role of flanking regions in establishing the amount of the solvent induced alpha-helical conformation and that of the splicedon in dictating the thermodynamic stability of its 'conformational trap' of the products, nonetheless one influences the other to some degree. We suggest that the stability of the 'conformational trap' of the product reflects the ability of the splicedon to 'recruit' the product conformation to protect the spliced peptide bond, i.e. to reduce the helix-coil transition of the spliced region which in turn imparts a degree of resistance to the spliced peptide bond.