Sunday, May 18, 2025
5/18/2025
Protein structure and dynamics in ultra-heterogeneous environments-Renewal - SUMMARY Hydrogen bonding in crowded environments – impact on biomolecules: Biomolecular organization in vivo is driven by crowding and heterogeneity. To date, protein structure, dynamics, and folding have been studied almost exclusively in buffer solutions, yet cellular environments are highly complex. This project seeks to characterize the driving forces behind protein structure and dynamics in environments that mimic the cytosol. Specifically: 1. We will quantify the sequence and crowder-dependent impact on the solvation shell of biomolecules, by mapping out the changes in solvent configurations and dynamics. 2. We will link changes in the microscopic H-bond environment with bulk effects on protein structure and stability. 3. We will advance 2D IR spectroscopic techniques to perform in-cell 2D IR measurements using labeled proteins in live mammalian cells to measure H-bond dynamics in the cytosol. We will directly address the question of “what are the properties of biological water?” which has been one of the key unresolved questions in the field. Liquid-liquid phase separation (LLPS) – biocondensate structure, dynamics, and interactions: Despite the popularity of biocondensates, the field has remained highly empirical. The residual secondary structure and stability of biocondensates, specifically the balance between electrostatic interactions and hydrogen bonding is not well understood. We seek to: 1. Investigate H-bond dynamics in partially disordered low-sequence- complexity condensates. We will induce LLPS using fusing Cryprochrome-2, a light-activated oligomerization protein to disordered domains (RGG) to generate “strong” (high physical crosslinking) and “weak” (low crosslinking) condensates in situ by UV illumination. We will probe the role of local interactions by comparing 2D IR measurements when LLPS is induced through crosslinking, crowding, or electrostatic interactions using RNA oligos. 2. We will investigate residual secondary structure in intrinsically disordered domains. Specifically, does LLPS promote folding. What is the role of LLPS in stabilizing secondary structure? Rational design of biologics – protein-polymer interactions: Proteins represent a rapidly emerging approach to therapeutics, though rapid degradation and low bioavailability pose a significant challenge. Polymer conjugation (PEGylation) is used to stabilize the proteins and increase bioavailability. However, most of the knowledge is empirical, as molecular models to explain how polymers interact with the protein surface are lacking. We will address the following hypotheses: 1. PEG-protein interactions are highly dynamic without specific “long-lived” contacts. Surface dehydration, or “water replacement” alters the backbone structure and dynamics. 2. Polymers modify thermal denaturation pathways. 3. PEG allergies highlight the need to explore alternative polymer architectures to avoid immunogenicity. Here, we will explore a series of alternative compositions including linear and branched polymers.
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