Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble

Proceedings of the National Academy of Sciences of the United States of America
Levani ZandarashviliJunji Iwahara

Abstract

Although engineering of transcription factors and DNA-modifying enzymes has drawn substantial attention for artificial gene regulation and genome editing, most efforts focus on affinity and specificity of the DNA-binding proteins, typically overlooking the kinetic properties of these proteins. However, a simplistic pursuit of high affinity can lead to kinetically deficient proteins that spend too much time at nonspecific sites before reaching their targets on DNA. We demonstrate that structural dynamic knowledge of the DNA-scanning process allows for kinetically and thermodynamically balanced engineering of DNA-binding proteins. Our current study of the zinc-finger protein Egr-1 (also known as Zif268) and its nuclease derivatives reveals kinetic and thermodynamic roles of the dynamic conformational equilibrium between two modes during the DNA-scanning process: one mode suitable for search and the other for recognition. By mutagenesis, we were able to shift this equilibrium, as confirmed by NMR spectroscopy. Using fluorescence and biochemical assays as well as computational simulations, we analyzed how the shifts of the conformational equilibrium influence binding affinity, target search kinetics, and efficiency in displacing ot...Continue Reading

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Oct 6, 2015·Biomolecules·Junji IwaharaLevani Zandarashvili
Feb 18, 2016·The Journal of Physical Chemistry Letters·Levani ZandarashviliJunji Iwahara
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