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Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore

Abstract

Here we describe a new type of biosensor element for detecting proteins in solution at nanomolar concentrations. We tethered a 3.4 kDa polyethylene glycol chain at a defined site within the lumen of the transmembrane protein pore formed by staphylococcal α-hemolysin. The free end of the polymer was covalently attached to a biotin molecule. On incorporation of the modified pore into a lipid bilayer, the biotinyl group moves from one side of the membrane to the other, and is detected by reversible capture with a mutant streptavidin. The capture events are observed as changes in ionic current passing through single pores in planar bilayers. Accordingly, the modified pore allows detection of a protein analyte at the single-molecule level, facilitating both quantification and identification through a distinctive current signature. The approach has higher time resolution compared with other kinetic measurements, such as those obtained by surface plasmon resonance.

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Figure 1: Preparation and analysis of heteroheptameric αHL pores.
Figure 2: Response of H6106C-PEG-biotin1 to WT and W120A streptavidins and a mouse anti-biotin mAb.
Figure 3: Kinetic model of the interactions between the αHL pore H6106C-PEG-biotin1 and streptavidin at the cis and trans sides of the bilayer.

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Acknowledgements

We are most grateful to Pat Stayton and David Hyre for the W120A protein. This work was supported by a Multidisciplinary University Research Initiative (MURI) award (Office of Naval Research) to H.B., a MURI award (Air Force Office of Scientific Research) to A.J. Welch, the Department of Energy and the Texas Advanced Technology Program. S.H. holds a postdoctoral fellowship from the Austrian Science Foundation (Fonds zur Förderung der wissenschaftlichen Forschung). We thank Li-Qun Gu, Yong Zhang, and Sean Conlan for their help and suggestions.

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Correspondence to Hagan Bayley.

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Movileanu, L., Howorka, S., Braha, O. et al. Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore. Nat Biotechnol 18, 1091–1095 (2000). https://doi.org/10.1038/80295

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