Proteins have been shown to be electrically-conductive if tethered to an electrode by means of a specific binding agent, allowing single molecules to be wired into an electrical sensing circuit. Such circuits allow enzymes to be used as as sensors, detectors and sequencing devices. We have engineered contact points into a Ф29 polymerase by introducing biotinylatable peptide sequences. The modified enzyme was bound to electrodes functionalized with streptavidin. Ф29 connected by one biotinylated contact and a second non-specific contact showed rapid small fluctuations in current when activated. Signals were greatly enhanced with two specific contacts. Features in the distributions of DC conductance increased by a factor 2 or more over the open-to closed conformational transition of the polymerase. Polymerase activity is manifested by rapid (millisecond) large (25% of background) current fluctuations imposed on the DC conductance.

Integrin Mac-1 (αMβ2) is an adhesion receptor vital to many functions of myeloid leukocytes. It is also the most promiscuous member of the integrin family capable of recognizing a broad range of ligands. In particular, its ligand-binding αMI-domain is known to bind cationic proteins/peptides depleted in acidic residues. This contradicts the canonical ligand-binding mechanism of αI-domains, which requires an acidic amino acid in the ligand to coordinate the divalent cation within the metal ion-dependent adhesion site (MIDAS) of αI-domains. The lack of acidic amino acids in the αMI-domain-binding sequences suggests the existence of an as-yet uncharacterized interaction mechanism. In the present study, we analyzed interactions of the αMI-domain with a representative Mac-1 ligand, the cationic cytokine pleiotrophin (PTN). Through NMR chemical shift perturbation analysis, cross saturation, NOESY, and mutagenesis studies, we found the interaction between the αMI-domain and PTN is divalent cation-independent and mediated mostly by hydrophobic contacts between the N-terminal domain of PTN and residues in the α5−β5 loop of αMI-domain. The observation that increased ionic strength weakens the interaction between the proteins indicates electrostatic forces may also play a significant role in the binding. On the basis of the results from these experiments, we formulated a model of the interaction between the αMI-domain and PTN.

Proteins have been shown to be electrically-conductive if tethered to an electrode by means of a specific binding agent, opening the possibility of building electronic devices that exploit the remarkable chemical versatility of enzymes by direct read-out of activity. Single-molecule conductance increases tenfold if two specific contacts are made (as is possible with bivalent antibodies). Here, we address the problem of forming contacts with proteins that, unlike antibodies, do not possess a multiplicity of native binding sites. In particular, we have engineered contact points into a Ф29 polymerase, with the points chosen so as to leave the active site free. Contacts are formed by introducing biotinylatable peptide sequences into Ф29 and functionalizing electrodes with streptavidin. Ф29 connected by one biotinylated contact and one non-specific contact shows rapid small fluctuations in current when activated. Signals are greatly enhanced with two specific contacts, a feature in the conductance distribution changing by > 9 nS between the open and closed conformations of the polymerase. Polymerase activity is accompanied by large changes in conductance over the millisecond timescale.