Revision as of 11:29, 17 October 2018

HOTag3 (Homo-Oligomeric Tag3)

Introduction

Some membrane-less organelles, such as stress granules and P bodies, have been discovered in recent years. Proteins condense into droplets and assemble these organelles through a process called phase separation. Physically, phase separation is the transformation of a one-phase thermodynamic system to a multi phase system, much like how oil and water demix from each other. According to thermodynamics, molecules will diffuse down the gradient of chemical potential instead of concentration. This is exactly why proteins will self organize into granules, diffusing from regions of low concentration to regions of high concentration. Here is an illustration of phase separation in cells.

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Design

When we wanted to rationally design a synthetic organelle based on phase separation and used it as a platform to achieve multi-functions, some design principles had to be followed. Interaction can bind the parts together while multivalence can make larger assemblies. In order to drive protein phase separation, we needed a multivalent module and a protein-protein interaction module. HOTag is the biobrick that we used to introduce multivalence. In natural process, such as phase separation occurred during T cell signal transduction, multivalency depends on multiple repeats protein domains. But it was not ideal to use multiple repeat domains in our design, because it would not only make the scaffold extremely large but also be problematic for molecular cloning and making transgenic yeasts. Thus, instead of using multiple repeats, we turned to de novo-designed homo-oligomeric short peptides. These short peptides are called HO-Tag (homo-oligomeric tag). HOTags contain approximately 30 amino acids. HOTag3 has high stoichiometry, forming hexamer spontaneously.

Figure 1. Structure of HOTags.

As for the protein-protein interaction part, we chose two sets of dimerization modules. The first pair was SUMO and SIM, which can dimerize spontaneously. The second one was chemically inducible FKBP and Frb. Rapamycin was the inducer of dimerization. We chose different modules according to the different functions we wanted to achieve. The hexameric HOTag3, together with another tetrameric HOTag (HOTag6), could robustly drive protein phase separation upon protein interaction (achieved by the protein-protein interaction module). Thus, HOTag3/6 pair is a useful tool to investigate protein phase separation and design a synthetic organelle. To verify the feasibility of the system, we fused two fluorescence proteins with the two components of synthetic organelles. We could observe the self-organization of components and the formation of organelles under fluorescence microscope. We named our system SPOT (Synthetic Phase separation-based Organelle Platform) because it could form granules (fluorescent spots) in yeast. Here is a demonstration of our overall design.

Figure 2. The overall design of the synthetic organelle with florescence reporters. SUMO-SIM module was fused with homo-oligomeric tags (HOTags) to form synthetic organelle.

Figure 3. FKBP-Frb module with homo-oligomeric tags (HOTags) to form synthetic organelle.

Properties

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Sequence and Features