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Published on: December 12, 2017
Electronic Circular Dichroism Detects Conformational Changes Associated with Proteasome Gating Confirmed Using AFM
Alessandro D'Urso1, Roberto Purrello1, Alessandra Cunsolo1
1Dipartimento Scienze Chimiche, Università degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy.
Abstract:
Many chronic diseases, including cancer and neurodegeneration, are linked to proteasome dysregulation. Proteasome activity, essential for maintaining proteostasis in a cell, is controlled by the gating mechanism and its underlying conformational transitions. Thus, developing effective methods to detect gate-related specific proteasome conformations could be a significant contribution to rational drug design. Since the structural analysis suggests that gate opening is associated with a decrease in the content of α-helices and β-sheets and an increase in random coil structures, we decided to explore the application of electronic circular dichroism (ECD) in the UV region to monitor the proteasome gating. A comparison of ECD spectra of wild type yeast 20S proteasome (predominantly closed) and an open-gate mutant (α3ΔN) revealed an increased intensity in the ECD band at 220 nm, which suggests increased contents of random coil and β-turn structures. This observation was further supported by evaluating ECD spectra of human 20S treated with low concentration of SDS, known as a gate-opening reagent. Next, to evaluate the power of ECD to probe a ligand-induced gate status, we treated the proteasome with H2T4, a tetracationic porphyrin that we showed previously to induce large-scale protein conformational changes upon binding to h20S. H2T4 caused a significant increase in the ECD band at 220 nm, interpreted as an induced opening of the 20S gate. In parallel, we imaged the gate-harboring alpha ring of the 20S with AFM, a technique that we used previously to visualize the predominantly closed gate in latent human or yeast 20S and the open gate in α3ΔN mutant. The results were convergent with the ECD data and showed a marked decrease in the content of closed-gate conformation in the H2T4-treated h20S. Our findings provide compelling support for the use of ECD measurements to conveniently monitor proteasome conformational changes related to gating phenomena. We predict that the observed association of spectroscopic and structural results will help with efficient design and characterization of exogenous proteasome regulators.
Insights
Electronic circular dichroism (ECD) can monitor proteasome gate conformational changes. This method detects changes related to disease and aids in designing new proteasome drugs.
Area of Science:
- Biochemistry
- Structural Biology
- Spectroscopy
Background:
- Proteasome dysregulation is linked to chronic diseases like cancer.
- Proteasome gating controls cellular proteostasis and is crucial for drug design.
- Understanding gate conformations is key for developing targeted therapies.
Purpose of the Study:
- To explore electronic circular dichroism (ECD) for monitoring proteasome gate conformations.
- To validate ECD as a tool for detecting structural changes associated with proteasome gating.
- To assess ECD's utility in characterizing ligand-induced proteasome gate status.
Main Methods:
- Utilized UV-region ECD spectroscopy to analyze wild-type yeast and human 20S proteasomes.
- Compared ECD spectra of closed and open-gate proteasome mutants.
- Applied ECD to monitor conformational changes induced by SDS and a tetracationic porphyrin (H2T4).
- Corroborated ECD findings with Atomic Force Microscopy (AFM) imaging of the proteasome alpha ring.
Main Results:
- ECD spectra showed distinct changes at 220 nm correlating with proteasome gate status (open vs. closed).
- Increased ECD intensity at 220 nm indicated higher random coil and beta-turn structures, associated with gate opening.
- H2T4 treatment induced a significant increase in the 220 nm ECD band, signifying induced gate opening.
- AFM results confirmed the ECD data, showing a decrease in closed-gate conformations upon H2T4 treatment.
Conclusions:
- ECD spectroscopy is a viable and convenient method for monitoring proteasome conformational changes related to gating.
- The correlation between ECD and structural data supports its application in drug design.
- This approach facilitates the characterization of exogenous proteasome regulators.
