Dramatic Structural and Thermodynamic Consequences of Repacking a Protein's Hydrophobic Core

Background: Rop is an RNA binding, dimeric, four-helix bundle protein with a well-defined, regular hydrophobic core ideally suited for redesign studies. A family of Rop variants in which the hydrophobic core was systematically redesigned has previously been created and characterized. Results: We present a structural and thermodynamic analysis of Ala(2)lle(2)-6, a variant of Rop with an extensively redesigned hydrophobic core. The structure of Ala(2)lle(2)-6 reveals a completely new fold formed by a conformational "flip" of the two protomers around the dimeric interface. The free-energy profile of Ala(2)lle(2)-6 is also very different from that of wild-type Rop. Ala(2)lle(2)-6 has a higher melting temperature than Rep, but undergoes a slightly smaller free-energy change on unfolding. Conclusions: The structure of Ala(2)lle(2)-6, along with molecular modeling results, demonstrate the importance of tight packing of core residues and the adoption of favorable core side chain rotamer values in determining helix-helix interactions in the four-helix bundle fold. Structural disorder at the N and C termini of Ala(2)lle(2)-6 provides a basis for the large differences in the enthalpy and entropy of Ala(2)lle(2)-6 folding compared with wildtype Rep.