This paper reports the discovery of a (meta)stable partially unfolded state of horse heart ferricytochrome c that was obtained after exposing the protein to a solution with an alkaline pH of 11.5 for 1 week. Thereafter, the protein did not undergo any detectable change in its secondary and tertiary structure upon adjusting the solution to folding promoting conditions at neutral pH. Spectroscopic data suggest that the misfolded protein exhibits a hexacoordinated low-spin state with a hydroxyl ion as the likely ligand. Below pH 6, a new ligation state emerges with the spectroscopic characteristics of a pentacoordinated quantum mixed state of the heme iron. Gel electrophoresis revealed substantial formation of soluble dimers and trimers at submillimolar concentrations, whereas monomers were dominant at lower, micromolar concentrations. Ultraviolet circular dichroism spectra indicate that oxidized monomers are pre-molten globule to globule-like with a substantial fraction of secondary (helical) structure reminiscent of alkaline state V. The oligomers contain even more helical structure, which suggests domain swapping as the underlying mechanism of their formation. A substantial fraction of the submillimolar mixture of monomers and oligomers underwent a reduction of the heme iron. Its dependence on pH suggests the coupling to a proton transfer process. Altogether, our data indicate a partially unfolded ferricytochrome c conformation with spectroscopic characteristics reminiscent of the recently discovered alkaline isomer V(b), which is stabilized under folding conditions by exposing the protein to a very alkaline pH for an extended period of time.
Natural proteins hide the essential requirements for their function beneath layers of unnecessary complexity. To test our understanding of structurefunction relationships and to develop proteins with novel properties, we engineer functional model proteins (maquettes) from scratch. Previously, we de-
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