NOE data demonstrating a compact unfolded state for an SH3 domain under non-denaturing conditions

The N-terminal SH3 domain of drk (drkN SH3) is unstable, existing in equilibrium between a folded state (F-exch) and an unfolded state (U-exch) under non-denaturing buffer conditions. Using a N-15/H-2-Iabeled sample, long range amide NOEs can be observed in the U-exch state as a result of reduced relaxation, in some cases correlating protons over 40 residues apart. These long range NOEs disappear upon addition of 2 M guanidinium chloride, demonstrating that there are substantial differences between the U-exch and the guanidine denatured states. Calculations using the long range NOEs of the U-exch state yield highly compact structures having non-native turns and a non-native buried tryptophan residue. These structures agree with experimental stopped-flow fluorescence data and analytical ultracentrifugation results. Since protein stability depends on the structural and dynamic properties of both the folded and unfolded states, this study provides insights into the stability of the drkN SH3 domain. These results provide the first strong NOE-based evidence for compact unfolded states of proteins and suggest that some unfolded states under physiological conditions have specific interactions leading to compact structures. (C) 1999 Academic Press.