Protein S is a calcium-binding protein comprising two Greek key beta-b
arrel domains. We have used NMR and optical spectroscopies to show tha
t, 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 sub
domains which are not contiguous along the polypeptide chain. The stru
ctures of these intermediates are incompatible with several previously
proposed folding mechanisms for Greek key beta-barrel domains. We pro
pose a different mechanism that involves multiple nucleation sites for
folding and sequential acquisition of native long-range interactions.
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