DIMER-TO-TETRAMER TRANSFORMATION - LOOP EXCISION DRAMATICALLY ALTERS STRUCTURE AND STABILITY OF THE ROP-4 ALPHA-HELIX BUNDLE PROTEIN

The ROP loop excision mutant RM6 shows dramatic changes in structure a nd stability in comparison to the wild-type protein. Removal of the fi ve amino acids (Asp30, Ala31, Asp32, Glu33, Gln34) from the loop resul ts in a complete reorganization of the protein as evidenced by single crystal X-ray analysis and thermodynamic unfolding studies. The homodi meric four-a-helix motif of the wild-type structure is given up. Inste ad a homotetrameric four-a-helix structure with extended, loop-free he lical monomers is formed. This intriguing structural change is associa ted with the acquisition of hyperthermophilic stability. This is evide nt in the shift in transition temperature from 71 degrees C characteri stic of the wild-type protein to 101 degrees C for RM6. Accordingly th e 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 transiti on proceeds highly cooperatively involving an enthalpy change of Delta H = 1073 +/- 30 kJ (mol of tetramer)(-1) and a heat capacity change a t the transition temperature of Delta(N)(D)C(p) = 14.9 (+/-) 3% kJ (mo l of tetramer x K)(-1). The two-state nature of the unfolding reaction is reflected in coinciding calorimetric and van't Hoff enthalpy value s. (C) 1998 Academic Press Limited.