| Recognition between molecules is a ubiquitous, fundamental process in biology and chemistry. Examples of recognition include enzyme and substrate binding, allosteric regulation, and transition-state stabilization. Understanding molecular recognition and how it can be altered remains a challenge. Knowing the structure of a protein is not sufficient; very subtle changes in orientation or motion can have significant functional consequences. In this dissertation protein engineering is used to manipulate molecular recognition to develop protein sensors and to explore adaptive evolution.; p53 was engineered to detect both proteases and antibodies by inserting peptides into the unstructured domains of p53 and generating variants that were activated up to 100-fold by the effectors chosen. In addition, an engineered p53 was incorporated into an existing high throughput screen for the detection of HIV protease. The mechanism of activation used to detect antibodies, induced dimerization, was used to develop a beta-glucuronidase sensor. Site-saturation mutagenesis and screening were used to generate an antibody-activated variant of beta-glucuronidase. The specific activity of the epitopetagged GUS variant was increased ∼500-fold by the addition of an equimolar concentration of a monoclonal antibody. These protein sensors are modular in design and could easily be re-engineered for the detection of other peptide-specific antibodies. Additionally, this dissertation reports the results of using beta-glucuronidase as a model enzyme for adaptive evolution by evolving it to have beta-xylosidase activity. In addition to evolving a beta-glucuronidase that had a 290,000-fold inversion of specificity, the results of experiments with the evolved enzyme show that saturation mutagenesis can be used to more rapidly adapt substrate specificity.; The results of this work demonstrate it is possible to use molecular recognition properties of proteins to change their function for use in a variety of ways, including the development of sensors. The sensors designed should have immediate utility in the detection of antibodies, which is commonly used to diagnose diseases. The sensors described are easier to use than current standards such as enzyme-linked immunosorbent assays. The success of our strategy with two very different proteins suggests that induced oligomerization could be a global way to convert any multimeric protein into a protein sensor. |
