Engineering of photocleavable protein (PhoCl) and light-inducible nanocapsules

• Author / Creator

  Lu, Xiaocen

• Optogenetics is a burgeoning range of biological techniques which involve the use of light and genetically encoded protein to control (actuators) or monitor (indicators) biochemical activities in living cells and tissues. In recent years, photo-controllable actuators engineered from photoreceptors have become widely used in various applications of chemical biology and physiology. In the optogenetic actuator toolkit, the key building block is a genetically encodable light-sensitive protein. Currently, most optogenetic actuators are engineered from naturally occurring photoreceptor protein from bacteria, fungi, and plants. Despite the tremendous progress in development and application of tools based on natural photoreceptors, the scope of physiological process that can be controlled remains limited by protein properties and photosensory mechanisms. To expand the actuator toolkit, the photocleavable protein (PhoCl), which is engineered from a photoconvertible protein, was introduced by our group in 2017. Illumination of PhoCl with violet light produces a large empty barrel fragment and a small chromophore-containing peptide fragment that dissociate spontaneously. Although the first generation of PhoCl (PhoCl1) has been demonstrated to be a simple and versatile photocleavable linker in a range of cell physiology and materials chemistry applications, an important drawback of the original PhoCl1 was a relative slow rate of dissociation, with a half-time of about 500 s. In this thesis, I describe my efforts in development of the second generation of PhoCl variants (PhoCl2) with improved rate and efficiency of dissociation. To better understand the structural changes associated with photoconversion and peptide dissociation, we determined the X-ray crystal structures of the green state, the red state, and the cleaved empty barrel of PhoCl1. Guided by the structural information, we further engineered PhoCl using a NanoLuc luciferase-based complementation assay for screening. Two variants of the second generation of PhoCl variants were developed via directed evolution. These variants were designated as PhoCl2c with higher dissociation contrast ratio and PhoCl2f with faster dissociation rate. Compared to the original PhoCl1, the PhoCl2 variants exhibited improved dissociation performances when characterized as purified proteins and in cell-based experiments. Furthermore, we describe the application of PhoCl2c for control of light-induced cell apoptosis in living cells. These data demonstrate that the new and improved variants, PhoCl2c and PhoCl2f, are useful for optogenetic control of subcellular localization and protein interactions. I also described the development of photo-inducible nanoparticles by engineering protein hybrids composed of encapsulin and a light-sensitive protein. Encapsulin is a virus capsid-like natural nanocapsule with diameter of 25 nm to 32 nm, which is used by bacteria to isolate toxic enzymatic activities. I demonstrated in mammalian cell expression system that enhanced green fluorescent protein (EGFP) can be displayed on the surface of the nanocapsules and a co-expressed cargo can be auto-packaged into the capsules in a fully genetically encodable way. To use encapsulin as a light-inducible capsule, I designed different constructs by fusing encapsulin to PhoCl or blue light photoactivable CRY2-CIBN system. We expect to employ this nanocapsule system for optogenetic release and caging of proteins involved in the control of in cell physiology.

• Subjects / Keywords

  • PhoCl
  • photocleavable protein
  • encapsulin

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-d1sw-pw14

• License

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