Research Projects

Biomolecular condensates are formed through liquid–liquid phase separation (LLPS). These are dynamic, membraneless compartments that are composed of proteins, RNA, or both. These condensates share features with synthetic coacervates, such as selective molecular partitioning and high local solute concentration, which make them promising candidates for catalysis in confined, water-based environments. However, most reported synthetic coacervates are derived from intrinsically disordered regions of sometimes disease-associated proteins, which makes them less desirable for medical application, limiting their broader applicability. This project aims to design and characterize a new class of biocompatible, peptide-based coacervates derived from the second prion domain (PRD2) domain of CPEB3 (Cytoplasmic Polyadenylation Element Binding Protein 3). CPEB3 is a functional prion involved in long-term memory formation. When the neurons are stimulated, CPEB3 is transformed from an inactive form (monomer) to an active aggregated form wherein they promote the activation of mRNA. These are blueprints that neurons use to manufacture other proteins involved in memory preservation. The aggregation of the CPEB3 proteins initiate the process through which memory traces at synapses are stabilized and preserved. Our preliminary work shows that PRD2 forms stable condensates and can catalyze organic reactions such as ester hydrolysis and redox transformations. We propose to leverage this system to develop reversible and tunable coacervates capable of acting as selective catalytic microenvironments under aqueous conditions. Many RNA binding proteins with similar domain composition as CPEB3 are shown to phase separate in cells. Homologues of CPEB3 in other organisms (like Orb2 in Drosophila) also form phase separated state. We hope to exploit the condensate forming CPEB family of protein to derive a set of minimal constructs that reversibly phase separate. The core hypothesis of our project is that the catalytic efficiency and selectivity of these coacervates can be tuned by altering their internal dynamics and molecular organization. We will engineer protein construct derived from CPEB3, its homologues, and from them the peptide variants via targeted sequence deletions and alterations that can efficiently phase seprate and form condensates. We will also be varying cofactors or incorporating binding partners for modifying the dynamics. These changes are expected to influence properties such as compartment fluidity, molecular exchange rates, and local microenvironment characteristics that impact catalysis. Nuclear Magnetic Resonance (NMR) spectroscopy will be the primary analytical tool, that we will employ to probe both structure and dynamics in these complex systems. NMR will allow us to simultaneously observe proteins, small molecules, and water to gain atomic-resolution insight into molecular partitioning, reaction intermediates, and structural organization within the coacervates. We will also use other biophysical and spectroscopic techniques for characterization. This includes, IR, Raman spectroscopy, fluorescence and FRAP techniques, zeta potential, conductivity measurements for the analysis. This integrative approach is expected to provide mechanistic understanding that can be directly translated into the rational design of improved coacervate-based catalysts. The result of the work will be critical for deeper fundamental understanding on how biocondensate influence enzyme catalysis. This will also help us in the development of biologically inspired catalytic systems, with potential applications in biotechnology, molecular engineering, and green chemistry. By advancing our understanding of condensate-mediated catalysis, the project addresses key challenges in sustainable chemistry by combining knowledge derived from soft matter, structural biology, and catalysis.