While we know a lot about the role of hydrophobic core packing in protein folding and stability, we still need more information about the role of other parts of a protein in its folding, stability, and activity. We have used a homodimer four-helix bundle protein (Rop) to investigate the role of its turn in the stability and activity of the protein. Rop’s tight turn has four amino acids (L29 D30 A31 D32) that connect two helices of a monomer. We made four libraries comprising all positions in the turn and selected active variants with a cell-based screen. Position 29 showed mostly medium-sized hydrophobic residues, and D30G showed up frequently in position 30, which is a known stabilizing mutation.  For position 31, the most abundant amino acid was surprisingly tryptophan. Also, although we hypothesized that a surface exposed ionic bond between D32 in the loop of a monomer and R55 of the other monomer may cause a bias in position 32 toward negatively charged amino acids, our libraries showed little bias in this position. In addition to measuring the thermal stability of selected library variants, we also rationally designed some variants to study the role of individual mutations in the overall stability and activity of the protein. We found although W is rarely seen in turns, A31W stabilizes Rop significantly. We also found that while most 5 amino acid turns in the library were somewhat destabilized, others such as one with a single Gly insertion are extremely stable. Rop’s turn tolerates a wide variety of amino acids, but it can play an important role in the stability of the protein. These results shed light on competing factors in turn and loop stability generally, as well as revealing some surprising routes to stabilization in Rop in particular.

Role of a four-helix bundle protein's loop in its stability and activity

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