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

Abstract

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 thiolreactive 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 cycloaddition (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.

N501Y mutation of spike protein in SARS-CoV-2 strengthens its binding to receptor ACE2

“…OaAEP1(C247A) is cysteine 247 to alanine mutant of asparaginyl endoprotease 1 from Oldenlandia affinis , abbreviated as Oa AEP1 here ( Yang et al, 2017 ; Shi et al, 2021 ). ELP is the elastin-like polypeptide ( Ott et al, 2017 ).…”

Interdomain Linker Effect on the Mechanical Stability of Ig Domains in Titin

“…A high-precision AFM measurement system has been used for accurate measurement and comparison of Ig domains in titin [ 64 , 65 , 66 , 67 ] ( Figure 1 d). In short, the target polyprotein designed with a specific peptide sequence NGL was immobilized on a GL peptide-coated surface through AEP (asparaginyl endopeptidase)-mediated protein ligation between the two peptide sequences [ 68 ]. A GB1-XDoc coated-AFM tip was used to probe the target polyprotein ( Figure 2 c).…”

Facile Synthesis of Peptide-Conjugated Gold Nanoclusters with Different Lengths

“…Enzymes can recognize many specific peptide sequences for ligation, such as sortase and asparaginyl ligase OaAEP1 [42,43,[55][56][57], and the use of a suitable peptide as a linker for protein immobilization may allow further characterization by single-molecule force spectroscopy [58][59][60][61][62][63][64][65][66], which may provide mechanical information about the nanocluster and immobilized protein [67][68][69][70][71]. Thus, by designing a proper peptide sequence for a first step peptide-conjugation, further functionalization and characterization of the nanoclusters can be possible [72][73][74][75][76][77].…”