McKenze J. Moss

Title: Probing Co-translational Folding Intermediates of a Kinetically Stable Protein

Proper folding is a prerequisite for protein function. Most proteins are multimeric, meaning that folding includes the additional step of assembling individual subunits together to form the native multimeric structure. Although little is known about multimeric protein folding, failing to form proper subunit interactions can lead to loss of protein function, aggregation and/or degradation. During protein synthesis, individual subunits of homomeric proteins are close to one other on neighboring ribosomes and may start to assemble co-translationally (“co-co” assembly). Alternatively, nascent chains may interact co-translationally with a full-length subunit (“co-post” assembly). Work from the Bukau and Kramer labs indicates that co-co interactions between nascent chains enhance the efficiency of homomeric protein folding and assembly (Bertolini et al. (2021) Science). Additionally, work from the Clark lab indicate that the co-translational folding pathway of the E. coli homotrimer chloramphenicol acetyltransferase (CAT) influences the final native structure (Walsh et al. (2020) PNAS). We are using CAT as a model system to understand how monomer folding and multimerization occurs co-translationally, during translation elongation. The CAT native trimer structure is thermostable to 80°C and shows no evidence of subunit exchange over the lifetime of E. coli. CAT does not refold to its native structure after dilution from a chemical denaturant, indicating that the “pioneer round” of CAT folding, potentially including co-translational folding and/or assembly, is particularly important for achieving the native CAT structure. However, we and others have shown that CAT native structure formation requires the presence of the CAT C-terminal residues (Van de Schueren et al. (1996) JMB). To probe CAT co-translational folding intermediates as a function of nascent chain length, we are using arrest-peptide force profiling coupled to a fluorescent reporter assay. This assay should prove useful for defining the in vivo folding pathway of CAT.

Dr. Trevor GrandPre

Title: Biophysical principles of condensates wetting membranes

Abstract:

Biomolecular condensates are intracellular compartments formed through phase separation of macromolecules. They often depend on interactions with membranes for processes such as localization, recruitment, and access to chemical substrates. These interactions are commonly facilitated by membrane-anchored tethers, a factor that is not considered in traditional wetting models. By employing a surface free-energy framework that integrates surface tension with tether density, we derive expressions for both the contact angle and tether density in a spherical cap configuration, extending the classic Young-Dupré equation. While the contact angle maintains its traditional force-balance interpretation, the tether density depends on the nature and strength of tether-condensate interactions. Using a straightforward interaction model, we analyze this relationship and obtain a wetting phase diagram that transitions from non-wetting to partial and complete wetting across a biologically relevant range of parameters. This study offers a quantitative approach to understanding condensate-membrane interactions and sheds light on how membranes may influence cellular organization and function.