Creating a Homeodomain with High Stability and DNA Binding Affinity by Sequence Averaging.
There is considerable interest in generating proteins with both high stability and high activity for biomedical and industrial purposes. One approach that has been used successfully to increase the stability of linear repeat proteins is consensus design. It is unclear the extent over which the consensus design approach can be used to produce folded and hyperstable proteins, and importantly, whether such stabilized proteins would retain function. Here we extend the consensus strategy to design a globular protein. We show that a consensus-designed homeodomain (HD) sequence adopts a cooperatively folded homeodomain structure. The unfolding free energy of the consensus-HD is 5 kcal·mol-1higher than that of the naturally occurring engrailed-HD from Drosophila melanogaster. Remarkably, the consensus-HD binds the engrailed-HD cognate DNA in a similar mode as the engrailed-HD with approximately 100-fold higher affinity.15N relaxation studies show a decrease in ps-ns backbone dynamics in the free state of consensus-HD, suggesting that increased affinity is not a result of increased plasticity. In addition to demonstrating the potential for consensus design of globular proteins with increased stability, these results demonstrate that greatly stabilized proteins can bind cognate substrates with increased affinities, showing that high stability is compatible with function.
The T. C. Jenkins Department of Biophysics and ‡Biomolecular NMR Center, Johns Hopkins University , 3400 North Charles Street, Baltimore, Maryland 21218, United States.
Katherine W
KW
Tripp
The T. C. Jenkins Department of Biophysics and ‡Biomolecular NMR Center, Johns Hopkins University , 3400 North Charles Street, Baltimore, Maryland 21218, United States.
Matt
M
Sternke
The T. C. Jenkins Department of Biophysics and ‡Biomolecular NMR Center, Johns Hopkins University , 3400 North Charles Street, Baltimore, Maryland 21218, United States.
Ananya
A
Majumdar
The T. C. Jenkins Department of Biophysics and ‡Biomolecular NMR Center, Johns Hopkins University , 3400 North Charles Street, Baltimore, Maryland 21218, United States.
Doug
http://orcid.org/0000-0001-7291-1389
D
Barrick