Combination of Click Chemistry and Enzymatic Ligation for Stable and Efficient Protein Immobilization for Single-Molecule Force Spectroscopy

Protein immobilization is an essential method for both basic and applied research for protein, and covalent, site-specific attachment is the most desirable strategy. Classic methods typically rely on a heterobifunctional cross-linker, such as N-hydroxysuccinimide (NHS)-linker-maleimide, or a similar two-step process. It utilizes the amino-reactive NHS and the thiol-reactive maleimide to conjugate protein to the solid support. However, NHS as a chemical is susceptible to hydrolysis during storage and handling, and maleimide reacts nonspecifically with all cysteines available in the protein, leading to an inconsistent result. To solve these problems, we have developed a method by combining a strain-promoted azide-alkyne cyclo-addition (SPAAC) click reaction and an OaAEP1 (C247A)-based enzymatic ligation. The method was demonstrated by the successful immobilization of enhanced green fluorescent protein (eGFP), which was visualized by fluorescent imaging. Moreover, the correct folding and stability of the immobilized protein were verified by atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) measurement with a high success rate (89%). Finally, the strength of the 1,2,3-triazole linkage from the azide-dibenzocyclooctyne (DBCO)-based SPAAC reaction was quantified with an ultrahigh rupture force >1.7 nN. Thus, this stable, efficient, and site-specific immobilization method can be used for many challenging systems, especially SMFS studies. [GRAPHICS] .

Automated summary

In this study, a protein immobilization method combining SPAAC click reaction and enzymatic ligation is developed to overcome the issues of NHS hydrolysis and nonspecific reaction of maleimide with the protein. The successful immobilization of eGFP and verification of its correct folding and stability demonstrate that this method is stable, efficient, and site-specific.