When attempting to master a new subject, it is usually advisable to seize its trunk, and avoid flailing about the branches. With proteins, however, most studies have focused on side chains, not backbones. Altering protein backbones has simply been beyond traditional methods of protein mutagenesis. And so it has been difficult to evaluate the importance of backbone-to-backbone hydrogen bonding in protein folding, protein stability, and protein-protein binding.
In recent years, however, biological and chemical techniques have been combined in novel ways to expose protein backbones to closer scrutiny. In particular, these techniques make it possible to insert amide-to-ester mutations in protein backbones, resulting in semisynthetic, or designer, proteins. Once created, these designer proteins can be examined systematically. It is even possible to evaluate the importance of individual hydrogen bonds for ligand binding.
A research group that is pushing semisynthetic proteins into new territory is based at the Center for Biopharmaceuticals at the University of Copenhagen. The group, which is led by Kristian Stromgaard, published their most recent study January 29 in Nature Communications, in an article entitled “Probing backbone hydrogen bonding in PDZ/ligand interactions by protein amide-to-ester mutations.” In this article, the authors describe how they generated semisynthetic PDZ domains containing backbone amide-to-ester mutations. PDZ domains are scaffolding modules in protein-protein interactions that mediate numerous physiological functions.
In other words, the researchers made miniscule changes in the backbone of in one of the most frequently occurring protein domains. Ultimately, they “created 22 completely new designer proteins on the basis of recognized material,” said Soren W. Pedersen, a postdoc in Stromgaard’s group. “We then examined how the designer proteins bind to other proteins in the body, which allowed us to analyze the role of the specific protein domain in the body's vital signal processes.”
The authors found “substantial and differential effects upon amide-to-ester mutation in PDZ2 of postsynaptic density protein 95 and other PDZ domains, suggesting that hydrogen bonding at the carboxylate-binding site contributes to both affinity and selectivity.” In addition, they noted that the hydrogen bonding pattern is surprisingly different between the canonical and noncanonical interactions.
Reflecting on the significance of these results, Pedersen commented, “Designer proteins [may] bind to a range of receptors in the body—receptor interactions that are important targets for pharmaceuticals intended to treat stroke, pain, and depression. The new findings mean that in the long term, we will be able to design pharmaceuticals that bind more strongly and more accurately to specific sites in the organism.”
Even more intriguingly, designer proteins may enable new receptor-interaction strategies. For example, rather than develop a pharmaceutical that affects a receptor directly, one may create one that alters the interactions that the receptor has with proteins inside the cell. In other words, instead of simply switching the function of the receptor on and off, one may effectively install a dimmer switch.