1.
Combinatorial Approaches for Efficient Design of Photoswitchable Protein-Protein Interactions as In Vivo Actuators.
Abstract:
Light switchable two-component protein dimerization systems offer versatile manipulation and dissection of cellular events in living systems. Over the past 20 years, the field has been driven by the discovery of photoreceptor-based interaction systems, the engineering of light-actuatable binder proteins, and the development of photoactivatable compounds as dimerization inducers. This perspective is to categorize mechanisms and design approaches of these dimerization systems, compare their advantages and limitations, and bridge them to emerging applications. Our goal is to identify new opportunities in combinatorial protein design that can address current engineering challenges and expand in vivo applications.
2.
Creating Red Light-Switchable Protein Dimerization Systems as Genetically Encoded Actuators with High Specificity.
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Huang, Z
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Li, Z
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Zhang, X
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Kang, S
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Dong, R
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Sun, L
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Fu, X
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Vaisar, D
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Watanabe, K
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Gu, L
Abstract:
Protein dimerization systems controlled by red light with increased tissue penetration depth are a highly needed tool for clinical applications such as cell and gene therapies. However, mammalian applications of existing red light-induced dimerization systems are hampered by limitations of their two components: a photosensory protein (or photoreceptor) which often requires a mammalian exogenous chromophore and a naturally occurring photoreceptor binding protein typically having a complex structure and nonideal binding properties. Here, we introduce an efficient, generalizable method (COMBINES-LID) for creating highly specific, reversible light-induced heterodimerization systems independent of any existing binders to a photoreceptor. It involves a two-step binder screen (phage display and yeast two-hybrid) of a combinatorial nanobody library to obtain binders that selectively engage a light-activated form of a photoswitchable protein or domain not the dark form. Proof-of-principle was provided by engineering nanobody-based, red light-induced dimerization (nanoReD) systems comprising a truncated bacterial phytochrome sensory module using a mammalian endogenous chromophore, biliverdin, and light-form specific nanobodies. Selected nanoReD systems were biochemically characterized, exhibiting low dark activity and high induction specificity, and further demonstrated for the reversible control of protein translocation and activation of gene expression in mice. Overall, COMBINES-LID opens new opportunities for creating genetically encoded actuators for the optical manipulation of biological processes.
