SUBDOMAIN INTERACTIONS AS A DETERMINANT IN THE FOLDING AND STABILITY OF T4 LYSOZYME

The folding of large, multidomain proteins involves the hierarchical a ssembly of individual domains. It remains unclear whether the stabilit y and folding of small, single-domain proteins occurs through a compar able assembly of small, autonomous folding units. We have investigated the relationship between two subdomains of the protein T4 lysozyme. T hermodynamically, T4 lysozyme behaves as a cooperative unit and the un folding transition fits a two-state model. The structure of the protei n, however, resembles a dumbbell with two potential subdomains: an N-t erminal subdomain (residues 13-75), and a C-terminal subdomain (residu es 76-164 and 1-12). To investigate the effect of uncoupling these two subdomains within the context of the native protein, we created two c ircular permutations, both at the subdomain interface (residues 13 and 75). Both variants adopt an active wild-type T4 lysozyme fold. The pr otein starting with residue 13 is 3 kcal/mol less stable than wild typ e, whereas the protein beginning at residue 75 is 9 kcal/mol less stab le, suggesting that the placement of the termini has a major effect on protein stability while minimally affecting the fold. When isolated a s protein fragments, the C-terminal subdomain folds into a marginally stable helical structure, whereas the N-terminal subdomain is predomin antly unfolded. ANS fluorescence studies indicate that, at low pH, the C-terminal subdomain adopts a loosely packed acid state. An acid stat e intermediate is also seen for all of the full-length variants. We pr opose that this acid state is comprised of an unfolded N-terminal subd omain and a loosely folded C-terminal subdomain.