Abstract
The mechanical properties of proteins can be studied with single molecule force spectroscopy (SMFS) using optical tweezers, atomic force microscopy and magnetic tweezers. It is common to utilize a flexible linker between the protein and trapped probe to exclude short-range interactions in SMFS experiments. One of the most prevalent linkers is DNA due to its well-defined properties, although attachment strategies between the DNA linker and protein or probe may vary. We will therefore provide a general overview of the currently existing non-covalent and covalent bioconjugation strategies to site-specifically conjugate DNA-linkers to the protein of interest. In the search for a standardized conjugation strategy, considerations include their mechanical properties in the context of SMFS, feasibility of site-directed labeling, labeling efficiency, and costs.
Highlights
Single molecule force spectroscopy (SMFS) has proven to be a powerful tool to investigate the properties of individual proteins, including mechanical stability [1], ligandbinding [2] and protein folding [3,4]
Background signals can be reduced by excluding non-specific short-range interactions between a surface and the protein of interest (POI) using a flexible linker between the protein and the trapped probe [5]
As only the mutation of a single amino acid is needed, the function of the POI has the potential to be less affected than when introducing a peptide-tag, functional tests should always be performed after mutation or insertion
Summary
Single molecule force spectroscopy (SMFS) has proven to be a powerful tool to investigate the properties of individual proteins, including mechanical stability [1], ligandbinding [2] and protein folding [3,4]. Digoxigenin would require fusion of the protein to the POI and are not practical The experimental for binding doApplications not require of additional catalysts and sufficient in conditions case of DNA-POI linking These handles are restricted to the of linkers to the probes. Strategies involving streptavidin and digoxigenin would require fusion of the protein to the POI and are not practical in case of DNA-POI linking Applications of these handles are restricted to the attachment of linkers to the probes. The two complexes provide a promising ligation strategy with extraordinarily strong non-covalent interactions, in practice it is a less elegant method; an additional method for the ligation of a protein (Dockerin or SdrG) to a DNA-linker is still required
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