Exploring the Folding Pathways of Annexin I, a Multidomain Protein. II. Hierarchy in Domain Folding Propensities may Govern the Folding Process Franc Ë oise Cordier-Ochsenbein 1 , Raphae È l Guerois 1 Franc Ë oise Russo-Marie 2 , Jean-Michel Neumann 1 and Alain Sanson 1 *{ 1 De Âpartement de Biologie Cellulaire et Mole Âculaire Section de Biophysique des Prote Âines et des Membranes and URA CNRS 2096, CEA Saclay, 91191 Gif sur Yvette Cedex, France 2 Institut Cochin de Ge Âne Âtique Mole Âculaire, Unite  INSERM U332, 22 rue Me Âchain 75014 Paris, France In the context of exploring the relationship between sequence and folding pathways, the multi-domain proteins of the annexin family constitute very attractive models. They are constituted of four 70-residue domains, named D1 to D4, with identical topologies but only limited sequence homology of approximately 30%. The domains are organized in a pseu- dochiral circular arrangement. Here, we report on the folding propensity of the D1 domain of annexin I obtained from overexpression in Escheri- chia coli. Unlike the D2 domain, which is only partially folded, the iso- lated D1 domain exhibits autonomous refolding in pure aqueous solution. Similarly, the D3 domain and D2-D3 module were obtained from expression in E. coli but were found to be largely unfolded. No con- clusion could be drawn for the D4 domain because it was not possible to extract it from the bacterial inclusion bodies. The data allow us to pro- pose a plausible scenario for the annexin I folding. This working model states that ®rstly the D1 domain folds, and the D2 and D3 domains remain partly unfolded, facilitating the docking of the D4 domain to the D1 domain. In a second step, the D1 and D4 domains dock, and D4 may fold if already not folded. The ®nal step starts with the stabilization of the D1-D4 module. This stabilization is crucial for allowing the non- native local interactions inside the still partially unfolded D2 domain to switch to the native long-range interactions involving D4. This switch allows the complete folding of D2 and D3. The model proposes a sequen- tial and hierarchical process for the folding of annexin I and emphasizes the role of both native framework and non-native structures in the process. # 1998 Academic Press Keywords: protein folding; NMR; annexin; stability; non-native structures *Corresponding author Introduction Understanding the process by which proteins reach their native structure is still a matter of deep and exciting research. While it is well known and clearly established that the three-dimensional structure of proteins is encoded in their sequence, the way these sequences also control the folding process is far from understood. The main reason for this is that there are very few proteins for which the folding process has been thoroughly analyzed as compared to the number of proteins for which the three-dimensional structure has been solved. A second important reason is prob- ably that proteins for which the folding process has been thoroughly studied and better under- {Also from Universite  P. et M. Curie, 9 Quai Saint- Bernard, Ba à t. C, 75005 Paris, France. F.C.-O. and R.G. contributed equally to this work and should be considered as joint ®rst authors. Abbreviations used: COSY, correlated spectroscopy; DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid; FPLC, fast performance liquid chromatography; GdnHCl, guanidinium choride; GST, glutathione-S-transferase; HMQC, heteronuclear multiple-quantum correlation; HSQC, heteronuclear single-quantum correlation; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; PCR, polymerase chain reaction; TFA, tri¯uoroacetic acid; TOCSY, total correlated spectroscopy. Article No. mb981828 J. Mol. Biol. (1998) 279, 1177±1185 0022 ± 2836/98/251177±09 $25.00/0 # 1998 Academic Press