Quantitative analysis of protein-protein interactions by native page/fluorimaging

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Nuclear transport is crucial for eukaryotic cell function.
• Importin proteins mediate the transport of proteins into the nucleus.
• Understanding protein-protein interactions in nuclear transport is key to cell biology.

Purpose of the Study:

• To develop a novel quantitative native PAGE mobility shift assay for measuring protein-protein binding affinities.
• To investigate the binding interaction between the Simian virus 40 (SV40) large tumour T-antigen (T-ag) nuclear localisation sequence (NLS) and importin (Imp) proteins.

Main Methods:

• Development of a quantitative native PAGE mobility shift assay.
• Utilizing a fluorescently labelled protein for detection.
• Analysis of the binding of SV40 T-ag NLS to importin alpha and the importin alpha/beta heterodimer.

Main Results:

• The developed assay can detect nanomolar (nM) binding affinities.
• SV40 T-ag NLS binds to the Imp alpha/beta heterodimer with an affinity of 340 nM.
• Binding to the Imp alpha/beta heterodimer is eight-fold higher than to Imp alpha alone, suggesting autoinhibition of Imp alpha.

Conclusions:

• The quantitative native PAGE mobility shift assay is a sensitive tool for analyzing protein-protein interactions in solution.
• The study elucidates the binding characteristics of SV40 T-ag NLS with importin proteins, providing insights into nuclear import mechanisms.
• The findings highlight the role of importin beta in enhancing the binding affinity of importin alpha to specific NLS sequences.