Rational design of p53, an intrinsically unstructured protein, for the fabrication of novel molecular sensors.
The dominant paradigm of protein engineering is structure-based site-directed mutagenesis. This rational approach is generally more effective for the engineering of local properties, such as substrate specificity, than global ones such as allostery. Previous workers have modified normally unregulated reporter enzymes, including beta-galactosidase, alkaline phosphatase, and beta-lactamase, so that the engineered versions are activated (up to 4-fold) by monoclonal antibodies. A reporter that could easily be "reprogrammed" for the facile detection of novel effectors (binding or modifying activities) would be useful in high throughput screens for directed evolution or drug discovery. Here we describe a straightforward and general solution to this potentially difficult design problem. The transcription factor p53 is normally regulated by a variety of post-translational modifications. The insertion of peptides into intrinsically unstructured domains of p53 generated variants that were activated up to 100-fold by novel effectors (proteases or antibodies). An engineered p53 was incorporated into an existing high throughput screen for the detection of human immunodeficiency virus protease, an arbitrarily chosen novel effector. These results suggest that the molecular recognition properties of intrinsically unstructured proteins are relatively easy to engineer and that the absence of crystal structures should not deter the rational engineering of this class of proteins.
Geddie
Melissa L
ML
Department of Biochemistry, Center for Fundamental and Applied Molecular Evolution, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
O'Loughlin
Taryn L
TL
Woods
Kristen K
KK
Matsumura
Ichiro
I
D000469:Alkaline Phosphatase / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001408:Bacillus anthracis / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D017930:Genes, Reporter; D014644:Genetic Variation; D005822:Genetic Vectors; D006678:HIV / Q000378:metabolism; D016333:HIV Protease / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D012323:RNA Processing, Post-Transcriptional; D013379:Substrate Specificity; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism / Q000502:physiology*; D001616:beta-Galactosidase / Q000378:metabolism; D001618:beta-Lactamases / Q000378:metabolism
D000911:Antibodies, Monoclonal; D010455:Peptides; D016159:Tumor Suppressor Protein p53; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase; D016333:HIV Protease; D001618:beta-Lactamases