Collapse of a Long Axis: Single-Molecule FörsterResonance Energy Transfer and Serpin Equilibrium Unfolding

摘要

The energy required for mechanical inhibition of target proteases is stored in the native structure of inhibitory serpins and accessed by serpin structural remodeling. The overall serpin fold is ellipsoidal with one long and two short axes. Most of the structural remodeling required for function occurs along the long axis, while expansion of the short axes is associated with misfolded, inactive forms. This suggests that ellipticity, as typified by the long axis, may be important for both function and folding. Placement of donor and acceptor fluorophores approximately along the long axis or one of the short axes allows single-pair Förster resonance energy transfer (spFRET) to report on both unfolding transitions and the time-averaged shape of different conformations. Equilibrium unfolding and refolding studies of the well-characterized inhibitory serpin α1-antitrypsin reveal that the long axis collapses in the folding intermediates while the monitored short axis expands. These energetically distinct intermediates are thus more spherical than the native state. Our spFRET studiesagree with other equilibrium unfolding studies that found that theregion around one of the β strands, s5A, which helps definethe long axis and must move for functionally required loop insertion,unfolds at low denaturant concentrations. This supports a connectionbetween functionally important structural lability and unfolding inthe inhibitory serpins.