Hossein Fazelinia
Shuang Yan Tang
Computational design of AraC protein with
novel effector specificity
Summary: Computer simulations play an increasingly significant role in understanding the underlying physical principles that dictate protein folding, stability and function, and computational advances in this area have greatly improved protein design predictions. In collaboration with Costas Maranas we are developing and testing a computational framework to rationally alter the effector binding specificity of the bacterial transcriptional regulatory protein AraC, belonging to the AraC/XylS family of transcriptional regulators.
Research in the Cirino laboratory is aimed at applying rational and combinatorial protein design methodologies to examine the capacity of genetic control components to adapt to new stimuli and effect varying levels of gene expression. Specifically, we would like to engineer bacterial transcriptional regulatory proteins that utilize specific, unnatural ligands as transcriptional activating "effector" molecules. The engineered proteins can then be used to couple these novel molecular recognition events to gene transcription, as demonstrated in recent work by Hellinga and coworkers
[1]. Our tailor-made transcriptional regulators will be used in metabolic engineering applications for the production of fine chemicals, and will contribute to the development of a genetic toolbox composed of standardized components, promoting a discipline of synthetic biology http://web.mit.edu/synbio/www
[2]. Regulatory proteins are being designed to activate transcription from their respective promoters only after a fine-tuned threshold concentration of a desired metabolite is reached. The ensuing gene transcription serves to report the presence/concentration of the target molecule through appropriate screening assays (e.g., via expression of a reporter protein) or selections (e.g, via expression of antibiotic resistance). Thus, as depicted in Figure 1, another objective here is to develop generally-applicable selection systems which will greatly facilitate directed evolution endeavors.
Alternatively, if the regulatory protein has been designed to respond to a molecule that is a pathway intermediate, the resulting gene regulation allows for tight control of downstream metabolic pathways, thereby eliminating unnecessary energy expenditures associated with continuous over-expression of heterologous enzymes [3,4]. Ultimately, several different engineered regulatory proteins could be incorporated into complex metabolic pathways containing branch points and used to initiate transcription from their corresponding promoters, depending on the metabolic state of the cell. This mechanism of control can be implemented in gene therapy as well, where gene expression must be tightly coupled to a cell's physiological state. Proteins which can selectively sense the presence of a target compound also have obvious implications in the design of clinical biosensors and other microanalytical devices. Finally, the technology developed here can also be extended to the design of protein therapeutics.