Role of contacts in long-range protein conductance

Abstract

Proteins are widely regarded as insulators, despite reports of electrical conductivity. Here we use measurements of single proteins between electrodes, in their natural aqueous environment to show that the factor controlling measured conductance is the nature of the electrical contact to the protein, and that specific ligands make highly selective electrical contacts. Using six proteins that lack known electrochemical activity, and measuring in a potential region where no ion current flows, we find characteristic peaks in the distributions of measured single-molecule conductances. These peaks depend on the contact chemistry, and hence, on the current path through the protein. In consequence, the measured conductance distribution is sensitive to changes in this path caused by ligand binding, as shown with streptavidin–biotin complexes. Measured conductances are on the order of nanosiemens over distances of many nanometers, orders of magnitude more than could be accounted for by electron tunneling. The current is dominated by contact resistance, so the conductance for a given path is independent of the distance between electrodes, as long as the contact points on the protein can span the gap between electrodes. While there is no currently known biological role for high electronic conductance, its dependence on specific contacts has important technological implications, because no current is observed at all without at least one strongly bonded contact, so direct electrical detection is a highly selective and label-free single-molecule detection method. We demonstrate single-molecule, highly specific, label- and background free-electronic detection of IgG antibodies to HIV and Ebola viruses.

Engineering an Enzyme for Direct Electrical Monitoring of Activity

“…The Gen I polymerase forms only one specific contact, but in contrast to what we have observed in the past for proteins with a single binding site, 3 there are two peaks in the conductance distribution, one at about 0.2 nS (characteristic of a weak, non-specific binding) and a second peak at ~ 1 nS. We speculate that the second peak is a consequence of additional surface linkages owing to the seven surface cysteines in this polymerase (conductance via a thiolated streptavidin is highly unlikely at this gap size - Figure S2).…”

“…Forward and reverse sweeps are superimposed. As observed for several other proteins, 3 the response is linear, with the exception of noise spikes that appear above ±100mV as a consequence of voltage-induced contact fluctuations. The slope of each trace was used to calculate a conductance, G=I/V and histograms of conductance (based on ~ 1000 measurements each) are shown for the three generations of polymerase in Figure 3.…”

“…This interpretation was confirmed using Fab fragments (one binding head) or functionalizing only one of the two electrodes with an epitope. 3 We also found that the internal conductance of proteins contacted via specific bindings agent is higher than the conductance associated with contact regions, so that that the measured electronic properties do not change much with gap size, until contact is lost with the protein. 3,4 Proteins are remarkably versatile molecular machines, capable of molecular recognition, highly selective catalysis, directional energy transfer, directed polymer synthesis and many other functions, so integration of proteins into bioelectronic devices has been a long sought-after goal, 5 and here we address the problem of making specific contacts to proteins that do not have multiple ligand binding sites, with the goal of demonstrating a device that transduces enzyme activity into electrical signals directly.…”

“…Furthermore, the internal conductance of the protein is sensitive to conformational changes. 3 Key to these experiments was the use of antibodies together with electrodes that were functionalized with an epitope to which the two binding domains of the antibody could bind. We measured the spread of conductances (owing to the range of contact geometries) by recording current-voltage (IV) curves from ~ 1000 molecules, and plotting histograms of the conductances derived from the IV curves.…”

“…We initially tested a Ф29 biotinylated just at the N terminus (Gen I), and bound to electrodes that had been covered with thiolated Streptavidin. Biotin-bound Streptavidin makes an excellent molecular wire 3 and also serves to keep the Ф29 (with its seven surface cysteines) away from the metal electrodes. We introduced a second contact about 5 nm distant from the first (Gen II), at a site in the (inactivated) exonuclease domain.…”