Journal of Biomolecular NMR, 19: 3–18, 2001. KLUWER/ESCOM © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 3 15 N NMR relaxation as a probe for helical intrinsic propensity: The case of the unfolded D2 domain of annexin I F. Ochsenbein ∗,∗∗ , R. Guerois ∗∗∗ , J.-M. Neumann ∗ , A. Sanson ∗ , E. Guittet & C. van Heijen- oort ∗∗∗∗ CNRS, Institut de Chimie des Substances Naturelles, 1 avenue de la terrasse, F-91190 Gif sur Yvette, France Received 7 July 2000; Accepted 13 November 2000 Key words: annexin, dynamics, 15 N NMR relaxation, protein folding, unfolded state Abstract The isolated D2 domain of annexin I is unable to adopt a tertiary fold but exhibits both native and non-native residual structures. It thus constitutes an attractive model for the investigation of dynamics of partially folded states in the context of protein folding and stability. 15 N relaxation parameters of the D2 domain have been acquired at three different magnetic fields, 500, 600 and 800 MHz. This enables the estimation of the contribution of conformational exchange to the relaxation parameters on the micro- to millisecond time scale, thus providing a suitable data set for the description of motions on the pico- and nanosecond time scale. The analysis of the seven spectral densities obtained (J (0), J (50 MHz), J (60 MHz), J (80 MHz), <J (500 MHz)>,<J (600 MHz)>, <J (800 MHz)>) provides complementary and meaningful results on the conformational features of the D2 domain structure previously depicted by chemical shift and NOE data. Especially, residual helix segments exhibit distinct dynamical behaviors that are related to their intrinsic helical propensity. Beside the spectral density analysis, a series of models derived from the Lipari and Szabo model-free approach are investigated. Two models containing three parameters are able to reproduce equally well the experimental data within experimental errors but provide different values of order parameters and correlation times. The inability to find a unique model to describe the data emphasizes the difficulty to use and interpret the model-free parameters in the case of partially or fully unfolded proteins consisting of a wide range of interconverting conformers. Abbreviations: CD, circular dichroism; CPMG, Carr, Purcell, Meiboom and Gill; CSA, chemical shift anisotropy; DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid; EDTA, ethylene-diamide-tetra-acetic acid; FID, free induction decay; MD, molecular dynamics; NOE, nuclear Overhauser effect; R N (N z ), longitudinal 15 N relaxation rate constant; R N (N x,y ), transverse 15 N relaxation rate constant; R N ( H N z ↔ N z ) , cross-relaxation rate between amide proton and nitrogen. Introduction Annexins constitute a family of multi-domain proteins characterized by a main C-terminal core whose three- ∗ Present address: D´ epartement de Biologie Cellulaire et Mol´ eculaire, Section de Biophysique des Prot´ eines et des Membranes, CEA de Saclay and URA CNRS 2096, F-91191 Gif sur Yvette Cedex, France. ∗∗ F. Ochsenbein and F. Cordier-Ochsenbein refer to the same author. ∗∗∗ Present address: Structural Biology program, EMBL, Meyer- hofstrasse 1, D-69117 Heidelberg, Germany. dimensional organization is highly hierarchical. This core consists of four ∼70-residue domains, D1 to D4 (Figure 1a), exhibiting an identical topology with only a limited sequence homology of approximately 30%. The domain topology comprises five helix segments, named A to E (Figure 1b), organized in a characteristic super-helix motif (Huber et al., 1992; Bewley et al., 1993; Concha et al., 1993; Weng et al., 1993). ∗∗∗∗ To whom correspondence should be addressed. E-mail: carine@icsn.cnrs-gif.fr