Dramatic structural and thermodynamic consequences of repacking a protein's hydrophobic core

Structure. 2000 Dec 15;8(12):1319-28. doi: 10.1016/s0969-2126(00)00544-x.


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 Ala2Ile2-6, a variant of Rop with an extensively redesigned hydrophobic core. The structure of Ala2Ile2-6 reveals a completely new fold formed by a conformational "flip" of the two protomers around the dimeric interface. The free-energy profile of Ala2Ile2-6 is also very different from that of wild-type Rop. Ala2Ile2-6 has a higher melting temperature than Rop, but undergoes a slightly smaller free-energy change on unfolding.

Conclusions: The structure of Ala2Ile2-6, 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 Ala2Ile2-6 provides a basis for the large differences in the enthalpy and entropy of Ala2Ile2-6 folding compared with wildtype Rop.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Bacterial Proteins / chemical synthesis
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacteriocin Plasmids / chemistry
  • Bacteriocin Plasmids / genetics
  • Circular Dichroism
  • Crystallization
  • Crystallography, X-Ray
  • Dimerization
  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Conformation
  • Protein Engineering
  • Protein Folding
  • Protein Isoforms / chemical synthesis
  • Protein Isoforms / chemistry
  • Protein Isoforms / genetics
  • Protein Structure, Secondary
  • RNA-Binding Proteins / chemical synthesis
  • RNA-Binding Proteins / chemistry*
  • RNA-Binding Proteins / genetics
  • Recombinant Proteins / chemical synthesis
  • Thermodynamics


  • Bacterial Proteins
  • Protein Isoforms
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Rop protein, ColE1 plasmid

Associated data

  • PDB/1F4M
  • PDB/1F4N