One-step asparaginyl endopeptidase (Oa AEP1)-based protein immobilization for single-molecule force spectroscopy

Abstract

Enzymatic protein ligation has become the most powerful and widely used method for high-precision atomic force microscopy single-molecule force spectroscopy (AFM-SMFS) study of protein mechanics. However, this methodology typically requires the functionalization of the glass surface with a corresponding peptide sequence/tag for enzymatic recognition and multiple steps are needed. Thus, it is time-consuming and a high level of experience is needed for reliable results. To solve this problem, we simplified the procedure using two strategies both based on asparaginyl endopeptidase (AEP). First, we designed a heterobifunctional peptide-based crosslinker, GL-peptide-propargylglycine, which links to an N ₃-functionalized surface via the click reaction. Then, the target protein with a C-terminal NGL sequence can be immobilized via the AEP-mediated ligation. Furthermore, we took advantage of the direct ligation between primary amino in a small molecule and protein with C-terminal NGL by AEP. Thus, the target protein can be immobilized on an amino-functionalized surface via AEP in one step. Both approaches were successfully applied to the AFM-SMFS study of eGFP, showing consistent single-molecule results.

Fig. 1. Scheme of surface protein immobilization using different methods. (A) First, the surface is functionalized by maleimide using the chemical crosslinker SMCC. Then, the target protein is immobilized via its endogenous cysteine. (B) First, the surface is functionalized by azide, and then maleimide is added using the crosslinker DBCO-linker-Mal. Then, the GL sequence is added using a peptide Cys-ELP-GL. Finally, the protein with NGL can be immobilized enzymatically. (C) a heterofunctional peptide with an alkyne group and a GL can be used for protein immobilization. (D) Target protein with a C-terminal NGL is ligated to the amino-functionalized glass surface in one step by AEP, and its molecular mechanism is depicted at the bottom.

Fig. 2. AFM-SMFS studies of immobilized eGFP. (A) Schemes show the heterofunctional peptide-based GFP immobilization for AFM-SMFS measurement. (B) Typical traces represent different unfolding pathways of GFP: one step (traces 1 and 2) and two steps (traces 3). (C) Scatter plots of F-ΔLc of the fused protein shows expected unfolding results for eGFP and GB1. (D) The histogram of the contour length of the construct shows a value of 82 nm.

Fig. 3. (A) The amino acid sequence of βMT shows eleven endogenous cysteines. (B) AFM-SMFS setup for βMT unfolding studies. (C and D) Representative force-extension curves and scatter plots of F-ΔLc of fused protein showed expected unfolding scenarios for each protein (domain).

Fig. 4. One-step immobilization of eGFP for AFM-SMFS study. (A) Scheme of AEP-mediate eGFP immobilization on the amino-functionalized glass surface. (B) Representative force-extension curves showing different unfolding pathways of GFP: one-step (traces 1 and 2) and two-step (trace 3). (C) Scatter plots of F-ΔLc of fused protein showed expected unfolding results for eGFP.

Peng Zheng