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Combining Rational Design and Continuous Evolution on Minimalist Proteins That Target DNA

preprint
submitted on 21.02.2020, 14:45 and posted on 24.02.2020, 06:00 by Jumi Shin, Ichiro Inamoto, Inder Sheoran, Serban Popa, Montdher Hussain
We designed MEF to mimic the basic region/helix-loop-helix/leucine zipper (bHLHZ) domain of transcription factors Max and Myc, which bind with high DNA sequence specificity and affinity to the E-box motif (enhancer box, CACGTG). To make MEF, we started with our rationally designed ME47, a hybrid of the Max basic region and E47 HLH, that effectively inhibited tumor growth in a mouse model of breast cancer. ME47, however, displays propensity for instability and misfolding. We therefore sought to improve ME47's structural and functional features. We used phage-assisted continuous evolution (PACE) to uncover "nonrational" changes to complement our rational design. PACE mutated Arg12 that contacts the DNA phosphodiester backbone. We would not have rationally made such a change, but this mutation improved ME47's stability with little change in DNA-binding function. We mutated Cys29 to Ser and Ala in ME47's HLH to eliminate undesired disulfide formation; these mutations reduced E-box binding activity. To compensate, we fused the designed FosW leucine zipper to ME47 to increase the dimerization interface and improve protein stability and E-box targeting activity. This "franken-protein" MEF comprises the Max basic region, E47 HLH, and FosW leucine zipper—plus mutations that arose during PACE and rational design—and is a tractable, reliable protein in vivo and in vitro. Compared with ME47, MEF gives three-fold stronger binding to Ebox with four-fold increased specificity for E-box over nonspecific DNA. Generation of MEF demonstrates that combining rational design and continuous evolution can be a powerful tool for designing proteins with robust structure and strong DNA-binding function.

Funding

NSERC, CHRP

History

Email Address of Submitting Author

jumi.shin@utoronto.ca

Institution

University of Toronto

Country

Canada

ORCID For Submitting Author

0000-0001-9939-2072

Declaration of Conflict of Interest

No conflict

Version Notes

submitted and now in review (as of last night) for JACS

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