Sequential protein expression and selective labeling for in-cell NMR in human cells

Area of Science:

• Biophysics
• Cell Biology
• Molecular Biology

Background:

• In-cell NMR offers atomic resolution insights into protein behavior within living human cells.
• Studying protein-protein interactions via NMR ideally requires selective isotopic labeling of only one protein partner.
• Current methods face challenges in achieving selective labeling within complex cellular environments.

Purpose of the Study:

• To develop and validate a strategy for selective isotopic labeling of a single protein in human cells.
• To enable the investigation of protein-protein interactions using in-cell NMR.
• To combine constitutive and transient protein expression with gene silencing for controlled labeling.

Main Methods:

• Established a human cell line stably overexpressing the copper binding protein HAH1.
• Utilized transient transfection to express and isotopically label human superoxide dismutase 1 (SOD1).
• Employed shRNA to silence HAH1 expression during SOD1 labeling and analyzed results using RT-PCR, Western Blot, and in-cell NMR.

Main Results:

• Achieved selective labeling of SOD1 within a time window of reduced HAH1 biosynthesis due to shRNA.
• In-cell NMR confirmed that only SOD1 was isotopically labeled and detectable, despite the presence of HAH1.
• Demonstrated successful suppression of HAH1 mRNA and protein levels via shRNA.

Conclusions:

• Controlling protein expression through targeted silencing of stably expressed proteins enables selective isotopic labeling.
• This technique facilitates the study of protein-protein interactions by in-cell NMR in human cells.
• The approach integrates established molecular biology techniques for broader applicability.