THE RELATIONSHIP BETWEEN STRUCTURE AND REACTIVITY IN RA-42, A DESIGNED HELIX-LOOP-HELIX MOTIF

RA-42, a polypeptide with 42 amino acids, has previously been shown to react with p-nitrophenyl fumarate (1) and site selectively form an am ide at the side chain of ornithine-15 in aqueous solution and in 10 vo l% 2,2,2-trifluoroethanol (TFE), In order to investigate the role of t he medium on structure and on reactivity the mean residue ellipticity at 222 nm has been measured by circular dichroism spectroscopy as a fu nction of TFE concentration and the second-order rate constant for the reaction between RA-42 and 1 has been measured in 30 vol% TFE, The co rrelation was poor between the mean residue ellipticity and the second -order rate constants, and the effect of increasing the TFE concentrat ion was therefore most likely to increase the strength of electrostati c interactions in the transition state due to the decreased polarity o f the solvent, The ratio of second-order rate constants between that o f RA-42 and that of 4-methylimidazole (4-MeIm), k(2)(RA-42)/k(2)(4-MeI m), increased with TFE concentration suggesting that there was little contribution to transition state binding in helix II since there is li ttle hairpin conformation left in 30 vol% TFE. Finally, the effect on structure of the introduced ornithines was determined by measuring the free energy of unfolding, Although the helix-loop-helix motif was des tabilised by the incorporation of positively charged residues, the eff ect on reactivity was small, suggesting that polypeptides are useful a s templates in the rational design of catalysts.