Abstract
Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.
Highlights
Protein-protein interactions are essential to most reactions in the cell and their characterization crucial for a better understanding of many fundamental processes in nature [1]
The complementary DNA strand is labeled with a Cy3 dye, forming a fluorescence resonance energy transfer (FRET) pair with the Cy5, as well as with a Biotin, which enables the coupling to the upper surface, a soft PDMS stamp functionalized with Streptavidin (Fig. 1A)
With the proof-of-principle system of nanobodies binding to Green Fluorescent Proteins (GFPs), we successfully demonstrated the implementation of the Molecular Force Assay in parallelized measurements of protein-protein interactions
Summary
Protein-protein interactions are essential to most reactions in the cell and their characterization crucial for a better understanding of many fundamental processes in nature [1]. We present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies.
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