Abstract
Electrical current recordings through electrolyte-filled nanopores (so called resistive pulse-sensing experiments) are attracting increasing attention for identifying and characterizing biomolecules. The majority of the work employing this method so far has focused on detection of oligonucleotides, polymers, and viruses. Most recently nanopores have been used to detect single proteins. This chapter reviews the very first attempts to use nanopores for characterizing properties of proteins that relate to their activity. The emphasis lies on those studies that provided quantitative information on activity-related properties of proteins, such as protein conformation, ligand binding, and enzyme activity. Nanopore-based studies have tremendous potential for investigating the function of proteins because the technique is capable of interrogating individual proteins at high-throughput without requiring labeling.
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Notes
- 1.
This adapted equation normalized ΔI with respect to the baseline current of translocation events, I. With this equation, the diameter of the molecules, d m , could be determined: \( {d_m}^3 = s\frac{{\Delta I}}{I}({l_p} + 0.8{d_p}){d_p}^2 \), with \( s \approx 1 \).
- 2.
The factor of 2 in the denominator of equation (9.2) is not present in the cited work by Talaga and Li. Working with Talaga and Li, we determined that the factor of 2 in the denominator is required for correct normalization such that the area of this probability density function equals 1.
- 3.
- 4.
Aptamers are short DNA or RNA segments that have been selected from a large pool (>10,000) of molecules and enriched using the SELEX technology. They often have high affinity for their target K d ~ 10−11.
- 5.
The catalytic rate constant k cat describes the rate at which the enzyme-substrate complex is converted to the free enzyme and free product. The Michaelis constant, K m , is the concentration of substrate that results in the half-maximal velocity of the enzymatic reaction.
- 6.
Gramicidin is a peptide consisting of 15 amino acids that spans one leaflet of a bilayer. If gA peptides are present in both leaflets of a bilayer, they can transiently form a dimer, which conducts monovalent cations through a central pore with diameter of ~4 Å. These transient ion channels result in discrete current values that reflect the number of ions passing through individual gA pores in a planar lipid bilayer at a given instant. Antonenko and coworkers first characterized the K d for the interaction of two monomers of gA that bind to form dimeric gA ion channels in a lipid bilayer [37].
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Acknowledgments
The authors acknowledge the following funding sources: National Institutes of Health (M.M., grant no. 1RO1GM081705), NSF Career Award (M.M., grant no. 0449088), AISIN/IMRA America Inc., and Thermo Fisher – CCG Collaborative Pilot Project Initiative.
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Yusko, E.C., Billeh, Y.N., Yang, J., Mayer, M. (2011). Nanopore Recordings to Quantify Activity-Related Properties of Proteins. In: Iqbal, S., Bashir, R. (eds) Nanopores. Springer, Boston, MA. https://doi.org/10.1007/978-1-4419-8252-0_9
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