http://hdl.handle.net/2374.MIA/5361 Dr. Richard Page - Associate Professor, Department of Chemistry and Biochemistry 2026-04-10T14:56:57Z http://hdl.handle.net/2374.MIA/7055 Data for: Functional and structural analyses of UbcH5 mutants with enhanced binding to the E3 ubiquitin ligase CHIP Manage, Maleesha; Page, Richard C Data describing structure-guided UbcH5b mutants that retain native activity profiles and structures while improving the affinity for CHIP. http://hdl.handle.net/2374.MIA/7052 Active Site Geometries for a Family of Metallo-beta-Lactamases Page, Richard; Wolke, Will Initial results from an analysis for a set of 84 metallo-beta-lactamase enzymes examining the internuclear distance between each zinc and each interacting atom, and the possible sets of angles formed by three atoms in which the central atom of the trio is one of the active site zinc ions. http://hdl.handle.net/2374.MIA/6904 Data: Real-Time Bio-Layer Interferometry Ubiquitination Assays as Alternatives to Western Blotting Page, Richard; DeSilva, Ruvindi Ubiquitination is a crucial cellular pathway enabling normal cellular functions such as cell cycle regulation, DNA damage repair, cell signaling, and maintenance of protein homeostasis. However, abnormalities or failures in the ubiquitination process can lead to cellular dysfunction and cause a range of diseases including cancers, neurodegenerative diseases, immune disorders, and metabolic disorders. Therefore, the ubiquitination cascade has become an attractive target for therapeutic interventions. However, screening and development of small molecule inhibitors that inhibit the activity of enzymes within the ubiquitination cascade is challenging due to the lack of knowledge of the complex dynamics and regulatory mechanisms of the cascade. Thus, the detection of ubiquitination is important for enabling a better understanding of the molecular mechanisms behind the ubiquitination cascade. Enormous efforts have been made in the field to detect ubiquitination using various techniques including fluorescence, spectrophotometry, chemiluminescence, NMR, and radioactive tracers. The most common method to detect ubiquitination is western blotting. However, western blotting is time-consuming and difficult to use when seeking fine-grained time course experiments. Here we present the use of bio-layer interferometry to rapidly assay ubiquitination in real-time. An E3 ligase auto-ubiquitination system and a substrate ubiquitination assay have been applied as tests for the newly developed assay. The developed BLI ubiquitination assay provides 1-second resolution and detects the formation of polyubiquitin chains directly on a biosensor-bound target. Results are returned instantaneously and result in substantial time savings. In addition, the reagent concentrations and quantities are similar to those used by traditional western blot-based ubiquitination assays. Thus, the developed BLI ubiquitination assay is introduced to the field as a viable alternative to traditional western blot assays to detect ubiquitination in a rapid real-time manner. http://hdl.handle.net/2374.MIA/6854 Modulation of Hsp70 Interactions with CHIP and HOP Co‐chaperones via Phosphorylation Page, Richard; Chathura, Paththamperuma Hsp70 plays a vital role in protein homeostasis. It associates with co-chaperones HOP and CHIP to promote protein refolding or degradative pathways, respectively. Phosphorylation of an Hsp70 threonine residue near the C-terminus modulates the binding affinity of Hsp70 towards co- chaperones HOP and CHIP. Upon Hsp70 C-terminal phosphorylation, Hsp70 preferentially binds with HOP as binding to CHIP is attenuated. Hsp70 is mainly found in the phosphorylated state in highly proliferating cells such as cancer, indicating upregulation of protein refolding over degradation. Unfortunately, the field lacks structural knowledge on how and why the affinity of Hsp70 for co-chaperones changes upon phosphorylation of the C-terminus. Here, we present structural and biophysical insights into the modulation of Hsp70 interactions with CHIP and HOP co-chaperones with phosphorylation at the C-terminus. Our data suggests that a rearrangement of the phosphorylated Hsp70 peptide and conformational changes induced in CHIP-TPR collectively lead to the disruption of an inter-molecular hydrogen bond in the complex of CHIP-TPR/pT-Hsp70 peptide. We built upon this structural knowledge to design novel CHIP-TPR variants to promote interaction of the CHIP-TPR with phosphorylated Hsp70. Our affinity data demonstrated that the novel variants exhibit higher affinity towards phosphorylated Hsp70 peptide compared to wild- type CHIP-TPR. We conducted molecular dynamics (MD) simulation studies to confirm effectiveness of the novel variants and suggest a mechanism by which the affinity is modulated. Our MD simulation data, in accordance with the crystallography data and the observed hydrogen bond network, explain the observed changes in binding affinity. Overall, this chapter explains the structural insights for modulation of the CHIP/Hsp70 interaction by phosphorylation status and the feasibility of designing biologically important CHIP variants to promote CHIP interactions with phosphorylated Hsp70.