STABILIZING AND DESTABILIZING EFFECTS OF PLACING BETA-BRANCHED AMINO-ACIDS IN PROTEIN ALPHA-HELICES

In order to gain greater insight into the effects of beta-branched ami no acids on protein alpha-helices, hydrophobic amino acids with varyin g degrees of beta-branching, including the fully beta-substituted L-2- amino-3,3-dimethylbutanoic acid (ADBA), were incorporated into the pro tein T4 lysozyme. The unnatural and natural amino acids were substitut ed at two solvent-exposed alpha-helical sites, Ser 44 and Asn 68, in t he protein using the technique of unnatural amino acid mutagenesis. Th e stabilities of the mutant proteins were determined by using a heat o f inactivation assay and from their circular dichroism thermal denatur ation curves. Surprisingly, while substitution of the amino acid with the greatest degree of beta-branching, ADBA, destabilizes the protein by 2.5 +/- 0.1 degrees C (0.69 +/- 0.03 kcal/mol) relative to Ala at s ite 44, the same substitution stabilizes the protein by 1.0 +/- 0.1 de grees C (0.27 +/- 0.03 kcal/mol) at site 68. The difference observed a t these two positions illustrates the extent to which the local contex t can mediate the impact of a particular mutation. Molecular dynamics simulations were carried out in parallel to model the structures of th e mutant proteins and to examine the energetic consequences of incorpo rating ADBA. Together, these results suggest that the conformationally restricted beta-branched amino acids are destabilizing, in part, beca use the beta-branched methyl groups can cause distortions in the local helix backbone. In addition, it is proposed that in some contexts the conformational rigidity of beta-branched amino acids may be stabilizi ng because it lowers the entropic cost of forming favorable side-chain van der Waals interactions.