A CONFORMATIONAL EQUILIBRIUM IN A PROTEIN-FRAGMENT CAUSED BY 2 CONSECUTIVE CAPPING BOXES - H-1-NMR, C-13-NMR, AND MUTATIONAL ANALYSIS

The conformational properties of an 18 residues peptide spanning the e ntire sequence, L(1)KTPA(5)QFDAD(10)ELRAA(15)MKG, of the first helix ( A-helix) of domain 2 of annexin I, were thoroughly investigated. This fragment exhibits several singular features, and in particular, two su ccessive potential capping boxes, T(3)xxQ(6) and D(8)xxE(11). The form er corresponds to the native hydrogen bond network stabilizing the alp ha helix N-terminus in the protein; the latter is a non-native capping box able to break the helix at residue D-8, and is observed in the do main 2 partially folded state. Using 2D-NMR techniques, we showed that two main populations of conformers coexist in aqueous solution. The f irst corresponds to a single helix extending from T-3 to K-17 The seco nd corresponds to a broken helix at residue D-8. Four mutants, T3A, F7 A, D8A, and E11A, were designed to further analyze the role of key ami no acids in the equilibrium between the two ensembles of conformers. T he sensitivity of NMR parameters to account for the variations in the populations of conformers was evaluated for each peptide. Our data sho w the delta(13)C(alpha) chemical shift to be the most relevant paramet er. We used it to estimate the population ratio in the various peptide s between the two main ensembles of conformers, the full helix and the broken helix. For the WT, E11A, and F7A peptides, these ratios are re spectively 35/65, 60/40, 60/40. Our results were compared to the data obtained from helix/coil transition algorithms.