EXPLORING THE FOLDING PATHWAYS OF ANNEXIN-I, A MULTIDOMAIN PROTEIN - I - NONNATIVE STRUCTURES STABILIZE THE PARTIALLY FOLDED STATE OF THE ISOLATED DOMAIN-2 OF ANNEXIN-I

Proteins of the annexin family constitute very attractive models becau se of their four similar to 70 residue domains, D1 to D4, exhibiting a n identical topology comprising five helix segments with only a limite d sequence homology of approximately 30%. We focus on the isolated D2 domain, which is only partially folded. A detailed analysis of this eq uilibrium partially folded state in aqueous solution and micellar solu tion using N-15-H-1 multidimensional NMR is presented. Comparison of t he residual structure of the entire domain with that of shorter fragme nts indicates the presence of long-range transient hydrophobic interac tions that slightly stabilize the secondary structure elements. The un folded domain tends to behave as a four-helix, rather than as a five-h elix domain. The ensemble of residual structures comprises: (i) a set of native structures consisting of three regions with large helix popu lations, in rather sharp correspondence with A, B and E helices, and a small helix population in the second part of the C helix; (ii) a set of non-native local structures corresponding to turn-like structures s tabilized by several side-chain to side-chain interactions and helix-d isruptive side-chains to backbone interactions. Remarkably, residues i nvolved in these local non-native interactions are also involved, in t he native structure, in structurally important non-local interactions. During the folding process of annexin I, the local non-native interac tions have to switch to native long-range interactions. This structura l switch reveals the existence of a sequence-encoded regulation of the folding pathways and kinetics, and emphasizes the key role of the non -native local structures in this regulation. (C) 1998 Academic Press.