NONNATIVE CAPPING STRUCTURE INITIATES HELIX FOLDING IN AN ANNEXIN-I FRAGMENT - A H-1-NMR CONFORMATIONAL STUDY

A 21-residue peptide, P(1)AQFD(5)ADELR(10)AAMKG(15)LGTDE(20)D, corresponding to the (helix A)-loop motif of the second repeat of human annexin I: was synthesized and studied by 2D proton NMR. The conformational properties of the peptide were characterized at different temperatures in pure aqueous solution and in a TFE/H2O (1:4 v/v) mixture. In pure aqueous solution, the peptide adopts a preferred conformation, comprising both elements of native and nonnative structures. A high alpha helix content is present in the DADELRA segment, which corresponds to an initiation site in the middle of the native alpha helix sequence. At the N-terminus flanking region, a particular nonnative folding is revealed by the J(NH-CH alpha) coupling constants and a set of unusual NOE connectivities which correspond to a helix interrupt at the first D residue. Addition of relatively small amount of TFE restores the native helix fold at the C-terminus but not at the N-terminus. On the contrary, the nonnative N-terminus structure is clearly stabilized by TFE. Our data indicate that this structure comprises (i) an Asps-x-x-Glu(8) N-terminal capping box, as recently named by Harper and Rose [Harper, E. T., & Rose, G. D. (1993) Biochemistry 32, 7605-7609], (ii) a (i,i+3) Asp(7)-x-x-Arg(10) salt bridge, and (iii) a hydrophobic cluster centered on Phe(4) which mainly interacts with Leu(9) but also with Ala(2) Ala(6), and Ala(12) in a dynamic way. This structure is rather stable since it is still observed at 293 K in aqueous solution and 313 K in the presence of TFE. It constitutes a very potent initiation site of the alpha helix structure. This is, however, a nonnative structure involving highly conserved residues in the whole annexin family and thus may play an important role in the folding pathway as a transient ''compacting helper''.