Identification of a stability determinant on the edge of the tet repressorfour-helix bundle dimerization motif

Isofunctional tetracycline repressor (TetR) proteins isolated from differen t bacteria show a sequence identity between 38 and 88% of the residues. The ir active state is a homodimer formed by a four-cr-helix bundle as the main interaction motif. We utilize this sequence variation of isofunctional pro teins to determine residues contributing to the stability of the four-helix bundle. The thermodynamic stabilities of two TetR proteins with 63% sequen ce identity were determined by urea-induced reversible denaturation followe d by fluorescence and circular dichroism. Both methods yield identical resu lts. The DeltaG degrees (U) (H2O) values are 60 and 75 kJ mol(-1). We have constructed TetR hybrid proteins derived from these wild types to identify the determinant leading to the 15 kJ mol(-1) stability difference. Successi ve size reduction of the exchanged portion yielded two single residues affe cting the overall protein stability. The P184Q exchange leads to a more sta ble protein, whereas the G181D exchange located at the solvent's exposed ed ge of the four-helix bundle is solely responsible for the reduced stability . Additional mutants based on crystal structures of TetR do not reveal any hint for steric interference of the Asp181 side chain with neighboring resi dues. Thus, this is an example for the role played by surface-exposed turn residues for the stability of four-helix bundles. We assume that the larger conformational flexibility of Gly and the reduction of the negative surfac e charge could favor formation of the turn on the edge of the four-helix bu ndle.