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 studie s. A family of Rop variants in which the hydrophobic core was systematicall y 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 s tructure of Ala(2)lle(2)-6 reveals a completely new fold formed by a confor mational "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 und ergoes 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 hel ix-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 diffe rences in the enthalpy and entropy of Ala(2)lle(2)-6 folding compared with wildtype Rep.