AN EXPERIMENTAL APPROACH TO EVALUATING THE ROLE OF BACKBONE INTERACTIONS IN PROTEINS USING UNNATURAL AMINO-ACID MUTAGENESIS

The contribution of backbone hydrogen bonds in a-helices to the overal l stability of a protein has been examined experimentally by replacing several backbone amide linkages in alpha-helix 39-50 of T4 lysozyme w ith ester linkages. T4 lysozyme variants wherein the backbone amide bo nds between residues Ser(38) and Leu(39), Lys(43) and Leu(44), or Ala( 49) and Ile(50) an replaced with ester bonds were generated by incorpo rating alpha-hydroxy acids at positions 39, 44, or 50, respectively, u sing unnatural amino acid mutagenesis. The stabilities of the proteins were determined from their thermal denaturation curves as monitored b y circular dichroism. Comparison of the thermal stabilities of the ami de-and ester-containing proteins shows that the ester substitution has a similar thermodynamic effect at all three positions. At the N- and C-terminal positions, where only one hydrogen-bonding interaction is p erturbed, the ester substitution is destabilizing by 0.9 and 0.7 kcal/ mol, respectively. Introduction of the ester linkage in the middle of the helix, which alters two hydrogen-bonding interactions, destabilize s the protein by 1.7 kcal/mol. The values obtained from these ester to amide mutations are compared to the values from similar mutations tha t have been made in other secondary structures and bimolecular complex es.