CONTEXT DEPENDENCE OF PROTEIN SECONDARY STRUCTURE FORMATION - THE 3-DIMENSIONAL STRUCTURE AND STABILITY OF A HYBRID BETWEEN CHYMOTRYPSIN INHIBITOR 2 AND HELIX E FROM SUBTILISIN CARLSBERG

The loop region of chymotrypsin inhibitor 2 from barley has been emplo yed as a scaffold for testing the intrinsic propensity of a peptide fr agment to form a secondary structure. The helix formation of the nine amino acid residue segment Lys-Gln-Ala-Val-Asp-Asn-Ala-Tyr-Ala of heli x E from subtilisin Carlsberg has been studied by the construction of a hybrid consisting of chymotrypsin inhibitor 2 (CI2) where part of th e active loop has been replaced by the nonapeptide. An expression syst em for a truncated form of CI2 where the 19 structureless residues of the N-terminus have been removed and Leu20 replaced by methionyl was c onstructed from the entire 83-residue wild-type CI2 gene by polymerase chain reaction methodology. The gene encoding the hybrid was construc ted from the truncated inhibitor gene. The stability of the truncated inhibitor and of the hybrid toward guanidinium chloride denaturation w as examined. From these measurements, the energy of unfolding in pure water was extrapolated to 30.5 +/- 1.0 kJ/mol for the truncated inhibi tor and 10.9 +/- 0.3 kJ/mol for the hybrid. These energies show that t he stability of CI2 is unaffected by the N-terminal truncation but sev erely decreased by the loop mutations. The three-dimensional structure of the hybrid protein has been determined in solution by nuclear magn etic resonance spectroscopy using 893 distance restraints and 84 torsi onal angle restraints. The average root-mean-square deviation (rmsd) o f 15 structures compared to their geometrical average was 0.8 +/- 0.2 angstrom for heavy backbone atoms and 1.3 +/- 0.2 angstrom for all hea vy atoms. The inserted peptide segment does not form an alpha-helix in the new structural context whereas the structure of the CI2 scaffold turns out to be amazingly conserved.