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 equili brium 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 ran ge amide NOEs can be observed in the U-exch state as a result of reduced re laxation, in some cases correlating protons over 40 residues apart. These l ong range NOEs disappear upon addition of 2 M guanidinium chloride, demonst rating that there are substantial differences between the U-exch and the gu anidine denatured states. Calculations using the long range NOEs of the U-e xch 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 o f both the folded and unfolded states, this study provides insights into th e 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 interact ions leading to compact structures. (C) 1999 Academic Press.