Calculation of ensembles of structures representing the unfolded state of an SH3 domain

The N-terminal SH3 domain of drk (drkN SH3 domain) exists in equilibrium be tween a folded (F-exch) and an unfolded (U-exch) form under non-denaturing conditions. In order to further our previous descriptions of the U-exch sta te, we have developed a protocol for calculating ensembles of structures, b ased on experimental spectroscopic data, which broadly represent the unfold ed state. A large number of unfolding trajectories were generated, starting from the folded state structure of the protein, in order to provide a reas onable sampling of: the conformational space accessible to this sequence. U nfolded state ensembles have been "calculated" using a newly developed prog ram ENSEMBLE, which optimizes the population weights assigned to each struc ture based on experimental properties of the U-exch state. Pseudo-energy te rms for nuclear Overhauser effects, J-coupling constants, C-13 chemical shi fts, translational diffusion coefficients and tryptophan ring burial based on NMR and fluorescence data have been implemented. The population weight a ssignment procedure was performed for different starting ensembles. Small n umbers of structures (<60) dominate the final ensembles compared to the tot al number in the starting ensembles, suggesting that the drkN SH3 domain U- exch state can be described by a limited number of lower-energy conformatio ns. The calculated U-exch state ensembles are much more compact than a "ran dom coil" chain, with significant native-like residual structure observed, in particular, a sizable population of conformers having the n-src loop and distal <beta>-hairpin structures exist in the calculated U-exch state ense mbles, and Trp36 is involved in a large number of interactions, both native and non-native. (C) 2001 Academic Press.