Equilibrium folding intermediates of a Greek key beta-barrel protein

Abstract

Protein S is a calcium-binding protein comprising two Greek key beta-barrel domains. We have used NMR and optical spectroscopies to show that, in the absence of calcium, the N-terminal domain of protein S forms two equilibrium folding intermediates that are in slow exchange. The intermediates arise from differential calcium-dependent folding of subdomains which are not contiguous along the polypeptide chain. The structures of these intermediates are incompatible with several previously proposed folding mechanisms for Greek key beta-barrel domains. We proposed a different mechanism that involves multiple nucleation sites for folding and sequential acquisition of native long-range interactions.