Engineering Synthetic Biomolecular Condensates with Rationally-Designed Modular Repeat Proteins

Biomolecular condensates (BCs) are membraneless organelles recently recognised as crucial regulators of numerous cellular processes, essential for cellular fitness and dynamic adaptation to environmental changes. They facilitate spatiotemporal control in critical cellular functions, including signalling, transcriptional regulation, stress response, and degradation pathways. Formed through phase separation of biomolecules such as proteins and nucleic acids, BCs exhibit remarkable structural and functional plasticity, resulting in their versatile material states and functional diversity across various cell types. Advances in microscopic techniques and computational simulations have substantially deepened our understanding of the molecular grammar guiding condensate formation, prompting the development of synthetic condensate systems. These engineered systems offer valuable insights into condensate behaviour, facilitate the incorporation of novel biological functionalities, and open potential therapeutic avenues. Despite significant progress, the precise contributions and modulatory roles of folded protein domains within BCs remain inadequately understood. This thesis investigates the rational design and characterisation of synthetic condensates based on consensus tetratricopeptide repeat (CTPR) proteins, modular folded non-globular domains, fused with low-complexity domains (LCDs), intrinsically disordered domains that drive phase separation. By systematically engineering a library of LCD-CTPR constructs sharing identical LCD sequences, this thesis examines how modifications to the folded domain, including repeat length, surface residue composition, and loop insertions, affect condensate phase behaviour. Integrative in vitro, in cellulo and in silico analyses reveal that changes in structured CTPR domains significantly modulate multivalent interactions among LCD-CTPR proteins, impacting condensate stability, phase boundaries, and material properties. Multiple fluorescence-based methods—including fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), fusion kinetic assays, and flicker spectroscopy—were employed to systematically characterise condensate dynamics and physicochemical properties. Leveraging the intrinsic modularity of CTPRs, the study expands condensate functionality by incorporating specific target-binding loops into the scaffolds, enabling programmable client recruitment. Partitioning assays demonstrate affinity-dependent recruitment of diverse proteins into condensates, highlighting how client binding can substantially alter material properties, prompting transitions to gel-like states, thus informing the future design of related constructs. To bridge in vitro characterisation with biological relevance, engineered LCD-CTPR condensates were introduced into the cellular environment. Supported by complementary in silico simulations, cellular assays confirmed the predictable behaviour of these designed condensates within complex biological contexts. Overall, this thesis provides a comprehensive framework integrating systematic in vitro, in cellulo, and computational approaches, laying foundational principles for the next generation of synthetic CTPR BCs design.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Ng, Tin Long Chris

Advisors dc:contributor.advisor

• Kumita, Janet
• Itzhaki, Laura

• Biomolecular Condensates
• Liquid–liquid phase separation (LLPS)
• Consensus tetratricopeptide repeat proteins (CTPRs)
• CTPR
• Synthetic Biology
• Multivalency and modular design
• Protein phase behaviour
• Protein Biology
• Biochemistry
• Biophysics
• Fluorescence microscopy
• Multivalency
• Intrinsically disordered proteins
• Protein–protein interactions
• Sequence-encoded interactions

Licence

• https://www.repository.cam.ac.uk/bitstreams/89a6eb46-35b8-4856-ab09-116724851614/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.122431

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/391222