Protein Sensing with Engineered Protein Nanopores
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The use of nanopores is a powerful new frontier in single-molecule sciences. Nanopores have been used effectively in exploring various biophysical features of small polypeptides and proteins, such as their folding state and structure, ligand interactions, and enzymatic activity. In particular, the α-hemolysin (αHL) protein pore has been used extensively for the detection, characterization, and analysis of polypeptides because this protein nanopore is highly robust, versatile, and tractable under various experimental conditions. Inspired by the mechanisms of protein translocation across the outer membrane translocases of mitochondria, we have shown the ability to use nanopore-probe techniques in controlling a single protein using engineered αHL pores. Here, we provide a detailed protocol for the preparation of αHL protein nanopores. Moreover, we demonstrate that placing attractive electrostatic traps is instrumental in tackling single-molecule stochastic sensing of folded proteins.
Key wordsα-Hemolysin Nanopore Single-channel electrical recordings Protein engineering Biotechnology and biosensors
This work was supported in part by grants from the US National Science Foundation (DMR-1006332) and National Institutes of Health (R01 GM088403) to LM.
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