Long-Range Conductivity in Proteins Mediated by Aromatic Residues.

Single-molecule measurements show that many proteins, lacking any redox cofactors, nonetheless exhibit electrical conductance on the order of a nanosiemen over 10 nm distances, implying that electrons can transit an entire protein in less than a nanosecond when subject to a potential difference of less than 1 V. This is puzzling because, for fast transport (i.e., a free energy barrier of zero), the hopping rate is determined by the reorganization energy of approximately 0.8 eV, and this sets the time scale of a single hop to at least 1 µs. Furthermore, the Fermi energies of typical metal electrodes are far removed from the energies required for sequential oxidation and reduction of the aromatic residues of the protein, which should further reduce the hopping current. Here, we combine all-atom molecular dynamics (MD) simulations of non-redox-active proteins (consensus tetratricopeptide repeats) with an electron transfer theory to demonstrate a molecular mechanism that can account for the unexpectedly fast electron transport. According to our MD simulations, the reorganization energy produced by the energy shift on charging (the Stokes shift) is close to the conventional value of 0.8 eV. However, the non-ergodic sampling of molecular configurations by the protein results in reaction-reorganization energies, extracted directly from the distribution of the electrostatic energy fluctuations, that are only ∼0.2 eV, which is small enough to enable long-range conductivity, without invoking quantum coherent transport. Using the MD values of the reorganization energies, we calculate a current decay with distance that is in agreement with experiment.

Heavy Water Reduces the Electronic Conductance of Protein Wires via Deuteron Interactions with Aromatic Residues.

Proteins are versatile, self-assembling nanoelectronic components, but their hopping conductivity is expected to be influenced by solvent fluctuations. The role of the solvent was investigated by measuring the single molecule conductance of several proteins in both H2O and D2O. The conductance of a homologous series of protein wires decreases more rapidly with length in D2O, indicating a 6-fold decrease in carrier diffusion constant relative to the same protein in H2O. The effect was found to depend on the specific aromatic amino acid composition. A tryptophan zipper protein showed a decrease in conductance similar to that of the protein wires, whereas a phenylalanine zipper protein was insensitive to solvent changes. Tryptophan contains an indole amine, whereas the phenylalanine aromatic ring has no exchangeable protons, so the effect of heavy water on conductance is a consequence of specific D- or H-interactions with the aromatic residues.

Systematics and biogeography of the Old World fern genus Antrophyum.

Antrophyum is one of the largest genera of vittarioid ferns (Pteridaceae) and is most diverse in tropical Asia and the Pacific Islands, but also occurs in temperate Asia, Australia, tropical Africa and the Malagasy region. The only monographic study of Antrophyum was published more than a century ago and a modern assessment of its diversity is lacking. Here, we reconstructed a comprehensively sampled and robustly supported phylogeny for the genus based on four chloroplast markers using Bayesian inference, maximum likelihood and maximum parsimony analyses. We then explored the evolution of the genus from the perspectives of morphology, systematics and historical biogeography. We investigated nine critical morphological characters using a morphometric approach and reconstructed their evolution on the phylogeny. We describe four new species and provide new insight into species delimitation. We currently recognize 34 species for the genus and provide a key to identify them. The results of biogeographical analysis suggest that the distribution of extant species is largely shaped by both ancient and recent dispersal events.

Measuring conductance switching in single proteins using quantum tunneling.

Interpreting the electrical signatures of single proteins in electronic junctions has facilitated a better understanding of the intrinsic properties of proteins that are fundamental to chemical and biological processes. Often, this information is not accessible using ensemble and even single-molecule approaches. In addition, the fabrication of nanoscale single-protein junctions remains challenging as they often require sophisticated methods. We report on the fabrication of tunneling probes, direct measurement, and active control (switching) of single-protein conductance with an external field in solution. The probes allowed us to bridge a single streptavidin molecule to two independently addressable, biotin-terminated electrodes and measure single-protein tunneling response over long periods. We show that charge transport through the protein has multiple conductive pathways that depend on the magnitude of the applied bias. These findings open the door for the reliable fabrication of protein-based junctions and can enable their use in future protein-embedded bioelectronics applications.

Electronic Transport in Molecular Wires of Precisely Controlled Length Built from Modular Proteins.

DNA molecular wires have been studied extensively because of the ease with which molecules of controlled length and composition can be synthesized. The same has not been true for proteins. Here, we have synthesized and studied a series of consensus tetratricopeptide repeat (CTPR) proteins, spanning 4 to 20 nm in length, in increments of 4 nm. For lengths in excess of 6 nm, their conductance exceeds that of the canonical molecular wire, oligo(phenylene-ethylenene), because of the more gradual decay of conductance with length in the protein. We show that, while the conductance decay fits an exponential (characteristic of quantum tunneling) and not a linear increase of resistance with length (characteristic of hopping transport), it is also accounted for by a square-law dependence on length (characteristic of weakly driven hopping). Measurements of the energy dependence of the decay length rule out the quantum tunneling case. A resonance in the carrier injection energy shows that allowed states in the protein align with the Fermi energy of the electrodes. Both the energy of these states and the long-range of hopping suggest that the reorganization induced by hole formation is greatly reduced inside the protein. We outline a model for calculating the molecular-electronic properties of proteins.

Probing Bioelectronic Connections Using Streptavidin Molecules with Modified Valency.

As molecular electronic components, proteins are distinguished by a remarkably long electronic decay length (∼10 nm) together with high contact resistance and extreme sensitivity to the chemical details of the contact. As a consequence, the conductance of even a large bioelectronic assembly is largely controlled by the conductance of the contacts. Streptavidin is a versatile linker protein that can tether together biotinylated electrodes and biotinylated proteins but with an ambiguity about the contact geometry that arises from its four possible binding sites for biotin. Here, we use engineered streptavidin tetramers, selected to contain a defined ratio of active monomers to "dead" monomers so as to define the biotin binding sites. We find a strong dependence of conductance on the separation of the biotin molecules, consistent with a short-range tunneling interaction within the streptavidin and in contrast to the long-range transport observed inside larger proteins. Hexaglutamate tails label the active monomers, and the additional negative charge enhances conductance significantly. This effect is quantitatively accounted for by an electronic resonance in the protein conductance.

The emerging landscape of single-molecule protein sequencing technologies.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

Moving Electrons Purposefully through Single Molecules and Nanostructures: A Tribute to the Science of Professor Nongjian Tao (1963-2020).

Electrochemistry intersected nanoscience 25 years ago when it became possible to control the flow of electrons through single molecules and nanostructures. Many surprises and a wealth of understanding were generated by these experiments. Professor Nongjian Tao was among the pioneering scientists who created the methods and technologies for advancing this new frontier. Achieving a deeper understanding of charge transport in molecules and low-dimensional materials was the first priority of his experiments, but he also succeeded in discovering applications in chemical sensing and biosensing for these novel nanoscopic systems. In parallel with this work, the investigation of a range of phenomena using novel optical microscopic methods was a passion of his and his students. This article is a review and an appreciation of some of his many contributions with a view to the future.

Ubiquitous Electron Transport in Non-Electron Transfer Proteins.

Many proteins that have no known role in electron transfer processes are excellent electronic conductors. This surprising characteristic is not generally evident in bulk aggregates or crystals, or in isolated, solvated peptides, because the outer hydrophilic shell of the protein presents a barrier to charge injection. Ligands that penetrate this barrier make excellent electrical contacts, yielding conductivities on the order of a S/m. The Fermi Energy of metal electrodes is aligned with the energy of internal electronic states of the protein, as evidenced by resonant transmission peaks at about 0.3V on the Normal Hydrogen Electrode scale. This energy is about 0.7 V less than the oxidation potential of aromatic amino acids, indicating a large reduction in electrostatic reorganization energy losses in the interior of the proteins. Consistent with a possible biological role for this conductance, there is a strong dependence on protein conformation. Thus, direct measurement of conductance is a powerful new way to read out protein conformation in real time, opening the way to new types of single molecule sensors and sequencing devices.

Electronic Conductance Resonance in Non-Redox-Active Proteins.

Bioelectronics research has mainly focused on redox-active proteins because of their role in biological charge transport. In these proteins, electronic conductance is a maximum when electrons are injected at the known redox potential of the protein. It has been shown recently that many non-redox-active proteins are good electronic conductors, though the mechanism of conduction is not yet understood. Here, we report single-molecule measurements of the conductance of three non-redox-active proteins, maintained under potential control in solution, as a function of electron injection energy. All three proteins show a conductance resonance at a potential ∼0.7 V removed from the nearest oxidation potential of their constituent amino acids. If this shift reflects a reduction of reorganization energy in the interior of the protein, it would account for the long-range conductance observed when carriers are injected into the interior of a protein.

Engineering an Enzyme for Direct Electrical Monitoring of Activity.

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 sensors, detectors, and sequencing devices. We have engineered contact points into a Φ29 polymerase by introducing biotinylatable peptide sequences. The modified enzyme was bound to electrodes functionalized with streptavidin. Φ29 connected by one biotinylated contact, and a second nonspecific 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 a rapid (millisecond) large (25% of background) current fluctuations imposed on the DC conductance.

Single Molecule Identification and Quantification of Glycosaminoglycans Using Solid-State Nanopores.

Glycosaminoglycans (GAGs) are a class of polysaccharides with potent biological activities. Due to their complex and heterogeneous composition, varied charge, polydispersity, and presence of isobaric stereoisomers, the analysis of GAG samples poses considerable challenges to current analytical techniques. In the present study, we combined solid-state nanopores-a single molecule sensor with a support vector machine (SVM)-a machine learning algorithm for the analysis of GAGs. Our results indicate that the nanopore/SVM technique could distinguish between monodisperse fragments of heparin and chondroitin sulfate with high accuracy (>90%), allowing as low as 0.8% (w/w) of chondroitin sulfate impurities in a heparin sample to be detected. In addition, the nanopore/SVM technique distinguished between unfractionated heparin (UFH) and enoxaparin (low molecular weight heparin) with an accuracy of ∼94% on average. With a reference sample for calibration, a nanopore could achieve nanomolar sensitivity and a 5-Log dynamic range. We were able to quantify heparin with reasonable accuracy using multiple nanopores. Our studies demonstrate the potential of the nanopore/SVM technique to quantify and identify GAGs.

Electronic Decay Length in a Protein Molecule.

Antibodies have two identical binding domains and can therefore form a well-defined conducting bridge by binding a pair of electrodes functionalized with an epitope. The conductance measured between these two fixed points on the antibody does not change with the size of the electrode gap. A second conduction path is via one specific attachment to an epitope and a second nonspecific attachment to the surface of the antibody. In this case, the conductance does change with gap size, yielding an estimated electronic decay length >6 nm, long enough that it is not possible to distinguish between an exponential or a hyperbolic distance dependence. This decay length is substantially greater than that measured for hopping transport in an organic molecular wire.

Role of contacts in long-range protein conductance.

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.

SIR proteins create compact heterochromatin fibers.

Heterochromatin is a silenced chromatin region essential for maintaining genomic stability and driving developmental processes. The complicated structure and dynamics of heterochromatin have rendered it difficult to characterize. In budding yeast, heterochromatin assembly requires the SIR proteins-Sir3, believed to be the primary structural component of SIR heterochromatin, and the Sir2-4 complex, responsible for the targeted recruitment of SIR proteins and the deacetylation of lysine 16 of histone H4. Previously, we found that Sir3 binds but does not compact nucleosomal arrays. Here we reconstitute chromatin fibers with the complete complement of SIR proteins and use sedimentation velocity, molecular modeling, and atomic force microscopy to characterize the stoichiometry and conformation of SIR chromatin fibers. In contrast to fibers with Sir3 alone, our results demonstrate that SIR arrays are highly compact. Strikingly, the condensed structure of SIR heterochromatin fibers requires both the integrity of H4K16 and an interaction between Sir3 and Sir4. We propose a model in which a dimer of Sir3 bridges and stabilizes two adjacent nucleosomes, while a Sir2-4 heterotetramer interacts with Sir3 associated with a nucleosomal trimer, driving fiber compaction.

Recognition Tunneling of Canonical and Modified RNA Nucleotides for Their Identification with the Aid of Machine Learning.

In the present study, we demonstrate a tunneling nanogap technique to identify individual RNA nucleotides, which can be used as a mechanism to read the nucleobases for direct sequencing of RNA in a solid-state nanopore. The tunneling nanogap is composed of two electrodes separated by a distance of <3 nm and functionalized with a recognition molecule. When a chemical entity is captured in the gap, it generates electron tunneling currents, a process we call recognition tunneling (RT). Using RT nanogaps created in a scanning tunneling microscope (STM), we acquired the electron tunneling signals for the canonical and two modified RNA nucleotides. To call the individual RNA nucleotides from the RT data, we adopted a machine learning algorithm, support vector machine (SVM), for the data analysis. Through the SVM, we were able to identify the individual RNA nucleotides and distinguish them from their DNA counterparts with reasonably high accuracy. Since each RNA nucleoside contains a hydroxyl group at the 2'-position of its sugar ring in an RNA strand, it allows for the formation of a tunneling junction at a larger nanogap compared to the DNA nucleoside in a DNA strand, which lacks the 2' hydroxyl group. It also proves advantageous for the manufacture of RT devices. This study is a proof-of-principle demonstration for the development of an RT nanopore device for directly sequencing single RNA molecules, including those bearing modifications.

A Y-Shaped Three-Arm Structure for Probing Bivalent Interactions between Protein Receptor-Ligand Using AFM and SPR.

The goal of this research was to develop linkage chemistry for the study of bivalent interactions between a receptor and its ligand using atomic force microscopy (AFM) and surface plasmon resonance (SPR). We conceived a three-arm structure composed of flexible chains connected to a large rigid core with orthogonal functional groups at their ends for formation and attachment (or immobilization) of bivalent ligands. To demonstrate the principle, we chose the well-known biotin-streptavidin interaction as a model system. On the basis of a crystal structure of the biotin-streptavidin complex, we designed and synthesized a bisbiotin ligand to have a Y shape with two biotin motifs on its arms for binding and a functional group on its stem for immobilization or attachment, referred to as y-bisbiotin. First, we found that the y-bisbiotin ligand stabilized the streptavidin more than its monobiotin counterpart did in solution, which indicates that the bivalent interaction was synergistic. The y-bisbiotin was attached to AFM tips through a click reaction for the force measurement experiments, which showed that unbinding the bisbiotin from streptavidin needed twice the force of unbinding a monobiotin. For the SPR study, we added a ω-thiolated alkyl chain to y-bisbiotin for its incorporation into a monolayer. The SPR data indicated that the streptavidin dissociated from a mixed monolayer bearing y-bisbiotin much slower than from the one bearing monobiotin. This work demonstrates unique chemistry for the study of bivalent interactions using AFM and SPR.

Observation of Giant Conductance Fluctuations in a Protein.

Proteins are insulating molecular solids, yet even those containing easily reduced or oxidized centers can have single-molecule electronic conductances that are too large to account for with conventional transport theories. Here, we report the observation of remarkably high electronic conductance states in an electrochemically-inactive protein, the ~200 kD αVß3 extracelluar domain of human integrin. Large current pulses (up to nA) were observed for long durations (many ms, corresponding to many pC of charge transfer) at large gap (>5nm) distances in an STM when the protein was bound specifically by a small peptide ligand attached to the electrodes. The effect is greatly reduced when a homologous, weakly-binding protein (α4ß1) is used as a control. In order to overcome the limitations of the STM, the time- and voltage-dependence of the conductance were further explored using a fixed-gap (5 nm) tunneling junction device that was small enough to trap a single protein molecule at any one time. Transitions to a high conductance (~ nS) state were observed, the protein being "on" for times from ms to tenths of a second. The high-conductance states only occur above ~ 100mV applied bias, and thus are not an equilibrium property of the protein. Nanoamp two-level signals indicate the specific capture of a single molecule in an electrode gap functionalized with the ligand. This offers a new approach to label-free electronic detection of single protein molecules. Electronic structure calculations yield a distribution of energy level spacings that is consistent with a recently proposed quantum-critical state for proteins, in which small fluctuations can drive transitions between localized and band-like electronic states.

Universal Readers Based on Hydrogen Bonding or π-π Stacking for Identification of DNA Nucleotides in Electron Tunnel Junctions.

A reader molecule, which recognizes all the naturally occurring nucleobases in an electron tunnel junction, is required for sequencing DNA by a recognition tunneling (RT) technique, referred to as a universal reader. In the present study, we have designed a series of heterocyclic carboxamides based on hydrogen bonding and a large-sized pyrene ring based on a π-π stacking interaction as universal reader candidates. Each of these compounds was synthesized to bear a thiolated linker for attachment to metal electrodes and examined for their interactions with naturally occurring DNA nucleosides and nucleotides by 1H NMR, ESI-MS, computational calculations, and surface plasmon resonance. RT measurements were carried out in a scanning tunnel microscope. All of these molecules generated electrical signals with DNA nucleotides in tunneling junctions under physiological conditions (phosphate buffered aqueous solution, pH 7.4). Using a support vector machine as a tool for data analysis, we found that these candidates distinguished among naturally occurring DNA nucleotides with the accuracy of pyrene (by π-π stacking interactions) > azole carboxamides (by hydrogen-bonding interactions). In addition, the pyrene reader operated efficiently in a larger tunnel junction. However, the azole carboxamide could read abasic (AP) monophosphate, a product from spontaneous base hydrolysis or an intermediate of base excision repair. Thus, we envision that sequencing DNA using both π-π stacking and hydrogen-bonding-based universal readers in parallel should generate more comprehensive genome sequences than sequencing based on either reader molecule alone.