Browsing by Author "Zechiedrich, Lynn"

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    Combining Random Gene Fission and Rational Gene Fusion To Discover Near-Infrared Fluorescent Protein Fragments That Report on Protein–Protein Interactions
    (American Chemical Society, 2015) Pandey, Naresh; Nobles, Christopher L.; Zechiedrich, Lynn; Maresso, Anthony W.; Silberg, Jonathan J.
    Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a proteinﾖprotein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and proteinﾖprotein interactions within whole animals.
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    DNA supercoiling-induced shapes alter minicircle hydrodynamic properties
    (Oxford University Press, 2023) Waszkiewicz, Radost; Ranasinghe, Maduni; Fogg, Jonathan M; Catanese, Daniel J, Jr.; Ekiel-Jeżewska, Maria L; Lisicki, Maciej; Demeler, Borries; Zechiedrich, Lynn; Szymczak, Piotr
    DNA in cells is organized in negatively supercoiled loops. The resulting torsional and bending strain allows DNA to adopt a surprisingly wide variety of 3-D shapes. This interplay between negative supercoiling, looping, and shape influences how DNA is stored, replicated, transcribed, repaired, and likely every other aspect of DNA activity. To understand the consequences of negative supercoiling and curvature on the hydrodynamic properties of DNA, we submitted 336 bp and 672 bp DNA minicircles to analytical ultracentrifugation (AUC). We found that the diffusion coefficient, sedimentation coefficient, and the DNA hydrodynamic radius strongly depended on circularity, loop length, and degree of negative supercoiling. Because AUC cannot ascertain shape beyond degree of non-globularity, we applied linear elasticity theory to predict DNA shapes, and combined these with hydrodynamic calculations to interpret the AUC data, with reasonable agreement between theory and experiment. These complementary approaches, together with earlier electron cryotomography data, provide a framework for understanding and predicting the effects of supercoiling on the shape and hydrodynamic properties of DNA.