Date of Award

Summer 7-16-2021

Degree Type


Degree Name

Doctor of Philosophy (PhD)




Castañeda, Carlos A.


Ess1, Folded domains, Intrinsically disordered regions, Liquid–liquid phase separation, NMR spectroscopy, Ubiquilin

Subject Categories

Biochemistry | Biochemistry, Biophysics, and Structural Biology | Biophysics | Life Sciences | Molecular Biology


Protein-protein interactions (PPIs) play central roles in most biological processes. Studying PPIs is a fundamental step in understanding the molecular basis of cellular processes such as cell-cell contact, enzyme activity, and transient assembly of signaling complexes or cellular structures. In this work, we employed a combination of biophysical and biochemical methods to characterize PPIs, with a focus on interactions between structured domains and intrinsically disordered regions or peptide substrates.The subject of Chapter two is prolyl isomerase Ess1, which is an essential enzyme found in Saccharomyces cerevisiae. Ess1 regulates the transcription and co-transcriptional RNA processing by catalyzing the isomerization of serine-proline bonds within the carboxy-terminal domain (CTD) of RNA polymerase II. We confirmed that, unlike its human ortholog Pin1, Ess1 has a rigid linker between its substrate-binding WW and catalytic domains. The rigid linker enforces a distance constraint and requires a minimum substrate length for bivalent CTD binding at the WW and catalytic domains simultaneously (> 4 heptad CTD repeats). Our binding results suggest that the Ess1 WW domain anchors the proximal end of the CTD substrate during isomerization and that linker divergence between Ess1 and Pin1 may underlie evolution of substrate specificity. Chapter three studied the interdomain interactions that mediate the liquid-liquid phase separation (LLPS) of ubiquilin-2 (UBQLN2). UBQLN2 is a shuttle protein for the proteasome that functions by binding to proteasomal receptors and ubiquitinated substrates via its N-terminal ubiquitin-like (UBL) and C-terminal ubiquitin associated (UBA) domains, respectively. UBQLN2 forms liquid droplets in vitro via a biophysical mechanism called liquid-liquid phase separation. We found that UBQLN2 LLPS is mediated by its middle intrinsically disordered region (IDR). The two folded domains of UBQLN2, the UBL and UBA domains, asymmetrically modulate LLPS by interacting with the IDR. We characterized the LLPS-inhibiting UBL-IDR interaction to be relatively strong and involves the STI1-I, linkers, and the C-terminal LLPS-mediating regions of UBQLN2. In contrast, the LLPS-enhancing UBA-IDR interaction is much weaker and involves mainly a middle linker and the C-terminal regions of the IDR after the STI1-II. Our results support a model in which binding partners could regulate UBQLN2 LLPS and its LLPS-associated functions in cells by interacting with either the UBL or UBA domains of the protein. In chapter four, we characterized and compared the temperature-dependent LLPS behaviors of UBQLN1 and UBQLN2 C-terminal constructs. UBQLN1 exhibits upper critical solution temperature (UCST) phase transition behavior; the protein condenses into liquid-like droplets at low temperature and returns to one soluble phase at high temperature. In contrast, UBQLN2 exhibits upper and lower critical solution temperature (UCST+LCST) phase transition; liquid droplets form at intermediate temperature and dissipate at high and low temperatures. We compared the sequences of UBQLN1 and UBQLN2 and identified a small glycine-rich region with significant sequence disparity between the two proteins. The glycine-rich region in UBQLN1 is rich in polar amino acids, whereas the same region in UBQLN2 is rich in hydrophobic residues. Using a chimera construct of UBQLN1, our data suggest that the dissimilar amino acid compositions of the glycine-rich region likely give rise to the different temperature-dependent LLPS behaviors of UBQLN1 and UBQLN2.


Open Access