Page, RichardDr. Richard Page - Associate Professor, Department of Chemistry and Biochemistryhttp://hdl.handle.net/2374.MIA/53612024-03-29T12:28:02Z2024-03-29T12:28:02ZData: Real-Time Bio-Layer Interferometry Ubiquitination Assays as Alternatives to Western BlottingPage, RichardDeSilva, Ruvindihttp://hdl.handle.net/2374.MIA/69042023-05-30T14:59:17ZData: 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.
Modulation of Hsp70 Interactions with CHIP and HOP Co‐chaperones via PhosphorylationPage, RichardChathura, Paththamperumahttp://hdl.handle.net/2374.MIA/68542022-12-20T00:38:49ZModulation 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.
Raw Data for: Fluorescence Dequenching Assay for the Activity of TEV Protease (YABIO_11495)Page, RichardPaththamperuma, Chathurahttp://hdl.handle.net/2374.MIA/68482022-10-18T01:08:05ZRaw Data for: Fluorescence Dequenching Assay for the Activity of TEV Protease (YABIO_11495)
Page, Richard; Paththamperuma, Chathura
Tobacco etch virus (TEV) protease is a widely used protease for fusion tag cleavage. Despite its widespread usage, an assay to quickly and easily quantify its activity in laboratory settings is still lacking. Thus, researchers may encounter inefficient cleavage of the desired fusion proteins due to poor activity of a given TEV protease preparation. Here, we describe the development and implementation of a fluorescence dequenching-based assay to quantify TEV protease activity and assess kinetic parameters. The peptide substrate used in this assay consists of a C-terminal TAMRA fluorophore, an N-terminal fluorescein fluorophore, and the canonical TEV protease recognition sequence. The assay is based on a reduction of fluorescence quenching of fluorescein upon cleavage by TEV protease. The substrate peptide was studied spectroscopically to assess feasibility and to propose a plausible mechanism of the assay. The assay was optimized and applied to obtain rapid assessments of TEV protease activity in purified samples and crude lysate extracts. The kinetic data obtained from improved TEV protease variants were compared with a traditional SDS-PAGE assay. Finally, the assay was applied to determine the optimum pH for TEV protease. Further, the study found that the assay is a rapid and simple approach to quantify TEV protease activity. The findings of the assay on crude lysate extracts, activity assay of TEV protease variants, and assessment of optimum pH for TEV protease reactions demonstrate the robust utility of the assay.
NIH R35 Plots - 20220309Page, Richardhttp://hdl.handle.net/2374.MIA/68092022-03-09T13:17:29ZNIH R35 Plots - 20220309
Page, Richard
Three plots of data on the R35 grant program at NIH.