Histidine-Specific Bioconjugation for Single-Molecule Force Spectroscopy
Source abstract
Abstract
Atomic force microscopy (AFM) based single-molecule force spectroscopy (SMFS) is a powerful tool to study the mechanical properties of proteins. In these experiments, site-specific immobilization of proteins is critical, as the tether determines the direction and amplitude of forces applied to the protein of interest. However, existing methods are mainly based on thiol chemistry or specific protein tags, which cannot meet the need of many challenging experiments. Here, we developed a histidine-specific phosphorylation strategy to covalently anchor proteins to an AFM cantilever tip or the substrate via their histidine tag or surface-exposed histidine residues. The formed covalent linkage was mechanically stable with rupture forces of over 1.3 nN. This protein immobilization method considerably improved the pickup rate and data quality of SMFS experiments. We further demonstrated the use of this method to explore the pulling-direction-dependent mechanical stability of green fluorescent protein and the unfolding of the membrane protein archaerhodopsin-3.
Source: openalex_inverted_index · Quality: baseline
Research context
OpenAlex topics: Force Microscopy Techniques and Applications · Mechanical and Optical Resonators · Lipid Membrane Structure and Behavior
SciVX discovery tags are terminology-based. OpenAlex topics are source-provided classifications.
Affiliations
Collaborative Innovation Center of Advanced Microstructures, National Laboratory of Solid State Microstructure, Department of Physics, Nanjing University 22 Hankou Road, Nanjing 210093, People’s Republic of China
Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University 163 Xianlin Road, Nanjing 210023, People’s Republic of China
Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou 325001, People’s Republic of China
Institute of Advanced Materials and Flexible Electronics (IAMFE), School of Chemistry and Materials Science, Nanjing University of Information Science & Technology 219 Ningliu Road, Nanjing, 210044, People’s Republic of China
Jinan Microecological Biomedicine Shandong Laboratory, Jinan 250021, People’s Republic of China
Funding
Ministry of Science and Technology of the People's Republic of China · State Key Laboratory of Precision Measurement Technology and Instruments · National Natural Science Foundation of China
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