An Experimental Approach to Evaluating the Role of Backbone Interactions in Proteins Using Unnatural Amino Acid Mutagenesis

Abstract

The contribution of backbone hydrogen bonds in alpha-helices to the overall stability of a protein has been examined experimentally by replacing several backbone amide linkages in alpha-helix 39-50 of T4 lysozyme with ester linkages. T4 lysozyme variants wherein the backbone amide bonds between residues Ser38 and Leu39, Lys43 and Leu44, or Ala49 and Ile50 are replaced with ester bonds were generated by incorporating alpha-hydroxy acids at positions 39, 44, or 50, respectively, using unnatural amino acid mutagenesis. The stabilities of the proteins were determined from their thermal denaturation curves as monitored by circular dichroism. Comparison of the thermal stabilities of the amide- 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 perturbed, 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, destabilizes the protein by 1.7 kcal/mol. The values obtained from these ester to amide mutations are compared to the values from similar mutations that have been made in other secondary structures and bimolecular complexes.

Revisiting Catalysis by Chymotrypsin Family Serine Proteases Using Peptide Substrates and Inhibitors with Unnatural Main Chains

“…Similarly, replacement of the P2 amide-NH of peptide VII with an oxygen atom (peptide XII) increased the K I for trypsin by a factor of approximately 7. These increases in K I correspond to ⌬⌬G values of approximately 1.2 kcal/mol, which is consistent with the loss of a single important hydrogen bond and similar to previous studies of amide to ester replacements within an ␣-helix that destabilized T4 lysozyme by 0.7-0.9 kcal/mol (34) and within an antiparallel ␤-sheet that reduced the stability of staphylococcal nuclease by 1.5-2.5 kcal/ mol (35). These data strongly suggest that both the main chain amide-NH and carbonyl groups of the P3 glycine of peptide X form hydrogen bonds, presumably with the main chain amide-NH and carbonyl groups of Gly-216 of trypsin, that significantly enhance the inhibitory potency of the peptide.…”

“…Replacement of the amide bond with an ester bond in the context of a peptide is an established strategy for investigating the role of peptide main chain hydrogen bonding in biochemical interactions (26,27,(32)(33)(34)(35). Amide and ester bonds are very similar in terms of structure and conformational preferences.…”

Nicotinic pharmacophore: The pyridine N of nicotine and carbonyl of acetylcholine hydrogen bond across a subunit interface to a backbone NH

“…A well-established strategy for probing potential backbone hydrogen bonds is to replace the residue that contributes the hydrogen bond donor with its α-hydroxy analog (Fig. 1B) (24)(25)(26)(27)(28). This mutation converts a backbone amide to a backbone ester, a subtle change that impacts backbone hydrogen bonding in two ways.…”

Energetics of Base Pairs in B-DNA in Solution: An Appraisal of Potential Functions and Dielectric Treatments

“…The hydrogen bond strength in R-helices without any additional parametrization of MHLF was estimated to be -1 kcal/mol 52 consistent with some recent experiments. 53,54 Usage of this function for DNA counterion interactions and for mapping out B to Z-DNA conformational energy profiles and integration into JUMNA have already been reported previously. [34][35][36] The electrostatic contribution to the interaction energy with the sigmoidal function is amenable to expression in a more familiar form [55][56][57] as the sum of Coulomb and shielding terms (due to solvent) for each pair of interacting atoms.…”