Real-time shape approximation and fingerprinting of single proteins using a nanopore Erik C. Yusko 1† , Brandon R. Bruhn 1† , Olivia M. Eggenberger 1,2 , Jared Houghtaling 1,2 , Ryan C. Rollings 3 , Nathan C. Walsh 3 , Santoshi Nandivada 3 , Mariya Pindrus 4 , Adam R. Hall 5 , David Sept 1,6 , Jiali Li 3 , Devendra S. Kalonia 4 and Michael Mayer 1,2,7 * Established methods for characterizing proteins typically require physical or chemical modification steps or cannot be used to examine individual molecules in solution. Ionic current measurements through electrolyte-filled nanopores can characterize single native proteins in an aqueous environment, but currently offer only limited capabilities. Here we show that the zeptolitre sensing volume of bilayer-coated solid-state nanopores can be used to determine the approximate shape, volume, charge, rotational diffusion coefficient and dipole moment of individual proteins. To do this, we developed a theory for the quantitative understanding of modulations in ionic current that arise from the rotational dynamics of single proteins as they move through the electric field inside the nanopore. The approach allows us to measure the five parameters simultaneously, and we show that they can be used to identify, characterize and quantify proteins and protein complexes with potential implications for structural biology, proteomics, biomarker detection and routine protein analysis. M ethods to characterize and quantify unlabelled, folded pro- teins in aqueous environments and on a single-molecule level do not currently exist 1 . If available, such methods could enhance routine protein analysis, enabling rapid and sensitive biomarker detection 2 , and allowing the analysis of personal pro- teomes 3 . Furthermore, if these methods could provide low- resolution approximations of shape, volume and dipole moment, they could help to reveal the conformation of transient protein complexes or large assemblies that are not accessible by electron microscopy, NMR spectroscopy, X-ray crystallography or small- angle X-ray scattering 4 . Despite the pioneering work by Oncley 5 , dipole moment has mostly been neglected as a protein descriptor and existing methods for determining protein dipole moments are tedious and limited to ensemble measurements. The dipole moment could, however, provide a powerful dimension for label-free protein analy- sis since absolute values range from zero to several thousand Debye among different proteins and are not correlated with protein size or charge 6 . Furthermore, the pharmaceutical industry is increasingly recognizing the importance of dipole moment for antibody formu- lations 7 , in part because subcutaneous injection of highly concen- trated solutions of monoclonal antibodies (the fastest growing class of therapeutics) can be impractical due to high viscosity and aggregation resulting from dipole alignment 7–9 . Nanopores can be used to interrogate single proteins. The approach requires a single electrolyte-filled pore in a thin insulating membrane that connects two solutions and can serve as a conduit for ions and proteins (Fig. 1a) 10,11 . Electrodes connect the solutions on both sides of the membrane to a high-gain amplifier that applies a constant electric potential difference, while measuring the ionic current through the nanopore. This arrangement ensures that the applied voltage drops almost entirely within the pore, rendering this zone sensitive to transient changes in its ionic conductivity. Consequently, each protein that is driven electrophoretically through the pore displaces conductive electrolyte, distorts the electric field and reduces the ionic current through the pore 12,13 . If the volume of the electrolyte-filled pore is sufficiently small com- pared with the volume of the particle, then the change in ionic current due to the translocating particle is measurable and charac- terized by its magnitude, ΔI, and duration, t d (refs 12,14 15–17); this current signature is referred to as a resistive pulse. In addition to its sensitivity to conductivity changes, this small volume transi- ently separates single proteins from other macromolecules in solution. As we report here, this allows the rotational dynamics of individual proteins to be interrogated and interpreted based on time- and orientation-dependent modulations of ionic current (Fig. 1b–e, Supplementary Notes 1–6 and Supplementary Figs. 1–9). Several groups have recently considered, in qualitative terms, the effect of a protein’s 14,16,18–21 or nanoparticle’s 12,22 shape when analys- ing distributed ΔI signals 23 and also the effect of a protein’s dipole on its translocation through an α-hemolysin pore in the presence of an a.c. field 24 . We have now developed a quantitative understanding of the dependence of measured ΔI values on the volume, shape, dipole moment and rotational diffusion coefficient of a protein inside a cylindrical nanopore, which makes it possible to estimate these parameters from resistive pulses (Supplementary Fig. 10). With further improvements, the ability to analyse individual proteins should mean the approximate shape of the protein, or the other four parameters, can be determined in mixtures of proteins without puri- fication; existing methods for determining the shape or structure of proteins either require purified, concentrated, or crystallized protein samples or the protein dynamics cannot be examined. 1 Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan 48109, USA. 2 Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, CH-1700 Fribourg, Switzerland. 3 Department of Physics, University of Arkansas, Fayetteville, Arkansas 72701, USA. 4 Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269, USA. 5 Department of Biomedical Engineering and Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston Salem, North Carolina 27157, USA. 6 Center for Computational Medicine and Biology, University of Michigan, Ann Arbor, Michigan 48109, USA. 7 Biophysics Program, University of Michigan, Ann Arbor, Michigan 48109, USA. † These authors contributed equally to this work. *e-mail: michael.mayer@unifr.ch ARTICLES PUBLISHED ONLINE: 19 DECEMBER 2016 | DOI: 10.1038/NNANO.2016.267 NATURE NANOTECHNOLOGY | ADVANCE ONLINE PUBLICATION | www.nature.com/naturenanotechnology 1 © 2016 Macmillan Publishers Limited, part of Springer Nature. 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