Dimer-to-tetramer transformation: Loop excision dramatically alters structure and stability of the ROP four α-helix bundle protein

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 ΔH = 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 x K)^-1. The two-state nature of the unfolding reaction is reflected in coinciding calorimetric and van't Hoff enthalpy values.