Phytochromes are biliprotein photoreceptors which exist in two photointerconvertible forms - a red light absorbing Pr form and far-red light absorbing Pfr form. Substitution of their native linear tetrapyrrole (bilin) prosthetic group with an unnatural bilin analog was shown to yield strongly fluorescent adducts that can be reconstituted in living cells(1). These self-assembling fluorescent adducts, phytofluors, hold great promise for in vivo cell biological applications; however, unlike the green fluorescent protein (GFP), exogenous bilins are needed for phytofluor formation in cells. In the present study, a directed evolution approach was undertaken with the goal of creating mutant phytochromes with novel spectroscopic properties. Error-prone PCR was employed to generate point mutations at random positions within the PHY domain of the cyanobacterial phytochrome Cph1. We hypothesize that alterations in this domain will result in spectrally shifted and red/far-red fluorescent holophytochrome mutants 'locked' in either the Pr or Pfr form. Apophytochrome mutant libraries, expressed in strains of Escherichia coli engineered to synthesize different bilin precursors, were screened using digital imaging spectroscopy (DIS) and fluorimaging methodologies(2;3). A variety of mutants with altered absorption properties were identified. In vitro DNA shuffling is in progress to enrich the diversity of mutant phenotypes.
Conference Committee Involvement (1)
Genetically Engineered Fluorescent and Bioluminescent Probes