RoL: EAGER: DESYN-C3 Membraneless organelles by design: a biomimetic approach

Project: Research project

Project Details


RoL: EAGER: DESYN-C3 Membraneless organelles by design: a biomimetic approach RoL: EAGER: DESYN-C3 Membraneless organelles by design: a biomimetic approach Biological liquid liquid phase separation (LLPS) of proteins is emerging as a fundamental process in living cells, which underpins the formation of functional, non-membrane bound, liquid-like compartments involved in cell function and development, such as the nucleolus, Cajal bodies, nuclear speckles, germ granules, processing bodies, stress granules, and cell signaling compartments. These membraneless organelles are coherent structures that can compartmentalize and concentrate specific sets of molecules in a highly dynamic manner, while exchanging components with their microenvironments on a rapid timescale. Within the liquid droplet, proteins maintain (or even acquire) a folded structure, and conserve function such as specific protein recognition. Increasingly, these structures are shown to play important roles in biological processes, and also are being implicated in diseases caused by protein aggregation. We hypothesize that LLPS can be exploited to generate designed membraneless organelles capable of performing complex functions. In this EAGER application, we will test our hypothesis by building an organelle that brings together elements of an enzyme cascade, thus improving the overall catalytic activity. In the long term, we will characterize these membraneless organelles in vivo by expressing the modular proteins recombinantly, and visualizing the formation of LLPS droplets by microscopy. Further, we will extend this approach to enable enzymatic cascades that include artificial metalloenzymes, such as those designed in the Ghirlanda lab. Our proposal addresses the following specific aims: 1- To demonstrate enzyme functionality in LLPS droplets containing a single enzyme, horseradish peroxidase (HPR). The enzyme will be linked to a series of intrinsically disordered designed sequences, with charge distribution designed to accommodate HRP. The constructs will be evaluated for their ability to form LLPS droplets and maintain enzyme functionality in vitro and in vivo. 2- To utilize LLPS droplets to reconstitute enzyme cascades. Protein modules designed to drive LLPS will be linked to two multienzyme systems: Glucose oxidase (GOx)/Horseradish peroxidase (HPR), and formate dehydrogenase (FDH)/formaldehyde dehydrogenase (FaldDH)/alcohol dehydrogenase (ADH). We will assess the functionality of LLPS droplets containing the cascades, and compare it with solution-phase mixtures of the enzymes. Intellectual merit. If successful, this proposal will establish an innovative, simple method to generate organelles for synthetic biology, complementing existing methods such as encapsulation by protein shells or by vesicles. Further, we will gain insights on the mechanism of LLPS from a molecular and physical standpoint. Our team includes an expert in protein design (Ghirlanda), a physicist who co-developed innovative methods to characterize LPS and the underlying theory in intrinsically disordered proteins (Vaiana), and an expert in computational modelling (Heyden). Broader impacts. Synthetic biology offers a unique opportunity to galvanize the enthusiasm of high school students. Building on existing collaborations with local high schools, we will set up a synthetic biology effort as part of the iGEM competition. Students will be involved in developing protein tags capable of triggering LLPS; the tags will be distributed through iGEM and
Effective start/end date9/1/188/31/21


  • National Science Foundation (NSF): $300,000.00


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