title: Improving a designed photocontrolled DNA-binding protein.
abstract: Photocontrolled transcription factors could be powerful tools for probing the roles of transcriptional processes in a variety of settings. Previously, we designed a photocontrolled DNA-binding protein based on a fusion between the bZIP region of GCN4 and photoactive yellow protein from Halorhodospira halophila [Morgan, S. A., et al. (2010) J. Mol. Biol. 399, 94-112]. Here we report a structure-based attempt to improve the degree of photoswitching observed with this chimeric protein. Using computational design tools PoPMuSiC 2.0, Rosetta, Eris, and bCIPA, we identified a series of single- and multiple-point mutations that were expected to stabilize the folded dark state of the protein and thereby enhance the degree of photoswitching. While a number of these mutations, particularly those that introduced a hydrophobic residue at position 143, did significantly enhance dark-state protein stability as judged by urea denaturation studies, dark-state stability did not correlate directly with the degree of photoswitching. Instead, the influence of mutations on the degree of photoswitching was found to be related to their effects on the degree to which DNA binding slowed the pB to pG transition in the PYP photocycle. One mutant, K143F, caused an ∼10-fold slowing of the photocycle and also showed the largest difference in the apparent K(d) for DNA binding, 3.5-fold lower, upon irradiation. This change in the apparent K(d) causes a 12-fold enhancement in the fraction bound DNA upon irradiation due to the cooperativity of DNA binding by this family of proteins. The results highlight the strengths and weaknesses of current approaches to a practical problem in protein design and suggest strategies for further improvement of designed photocontrolled transcription factors.
authors.affiliation: Faculty of Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1.
authors.forename: Helen Y
authors.initials: HY
authors.lastname: Fan
authors.forename: Stacy-Anne
authors.initials: SA
authors.lastname: Morgan
authors.forename: Katherine E
authors.initials: KE
authors.lastname: Brechun
authors.forename: Yih-Yang
authors.initials: YY
authors.lastname: Chen
authors.forename: Anna S I
authors.initials: AS
authors.lastname: Jaikaran
authors.forename: G Andrew
authors.initials: GA
authors.lastname: Woolley
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000138:chemical synthesis* / Q000235:genetics; D020607:Halorhodospira halophila / Q000235:genetics / Q000378:metabolism; D008027:Light; D008164:Luminescent Proteins / Q000138:chemical synthesis* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D055668:Photochemical Processes; D015202:Protein Engineering / Q000379:methods*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D004247:DNA