Abstract
The ROP loop excision mutant RM6 shows dramatic changes in structure and stability in comparison to the wild-type protein. Removal of the five amino acids (Asp30, Ala31, Asp32, Glu33, Gln34) from the loop results in a complete reorganization of the protein as evidenced by single crystal X-ray analysis and thermodynamic unfolding studies. The homodimeric four-α-helix motif of the wild-type structure is given up. Instead a homotetrameric four-α-helix structure with extended, loop-free helical monomers is formed. This intriguing structural change is associated with the acquisition of hyperthermophilic stability. This is evident in the shift in transition temperature from 71°C characteristic of the wild-type protein to 101°C for RM6. Accordingly the Gibbs energy of unfolding is increased from 71.7 kJ (mol of dimer) −1 to 195.1 kJ (mol of tetramer) −1. The tetramer-to-monomer transition proceeds highly cooperatively involving an enthalpy change of Δ H=1073±30 kJ (mol of tetramer) −1 and a heat capacity change at the transition temperature of Δ N D C p=14.9(±)3% kJ (mol of tetramer × K) −1. The two-state nature of the unfolding reaction is reflected in coinciding calorimetric and van’t Hoff enthalpy values.
Published Version
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