Significant efforts have recently been invested in assessing the physical and chemical properties of microbial nanowires for their promising role in developing alternative renewable sources of electricity, bioelectronic materials and implantable sensors. One of their outstanding properties, the ever-desirable conductivity has been the focus of numerous studies. However, the lack of a straightforward and reliable method for measuring it seems to be responsible for the broad variability of the reported data. Routinely employed methods tend to underestimate or overestimate conductivity by several orders of magnitude. In this work, synthetic peptide nanowires conductivity is interrogated employing a non-destructive measurement technique developed on a terahertz scanning near-field microscope to test if peptide aromaticity leads to higher electrical conductivity. Our novel peptide conductivity measurement technique, based on triple standards calibration method, shows that in the case of two biopolymer mimicking peptides, the sample incorporating aromatic residues (W6) is about six times more conductive than the negative control (L6). To the best of our knowledge, this is the first report of a quantitative nano-scale terahertz s-SNOM investigation of peptides. These results prove the suitability of the terahertz radiation-based non-destructive approach in tandem with the designer peptides choice as model test subjects. This approach requires only simple sample preparation, avoids many of the pitfalls of typical contact-based conductivity measurement techniques and could help understanding fundamental aspects of nature's design of electron transfer in biopolymers.
The filamentous peptide-based nanowires produced by some dissimilatory metal-reducing bacteria, such as Geobacter sulfurreducens, display excellent natural conductivity. Their mechanism of conduction is assumed to be a combination of delocalized electrons through closely aligned aromatic amino acids and hopping/charge transfer. The proteins that form these microbial nanowires are structured from a coiled-coil, for which the design rules have been reported in the literature. Furthermore, at least one biomimetic system using related synthetic peptides has shown that the incorporation of aromatic residues can be used to enhance conductivity of peptide fibers. Herein, the de novo design of peptide sequences is used to enhance the conductivity of peptide gels, as inspired by microbial nanowires. A critical factor hampering investigations in both microbiology and materials development is inconsistent reporting of biomaterial conductivity measurements, with consistent methodologies needed for such investigations. We have reported a method herein to analyze non-Ohmic behavior using existing parameters, which is a statistically insightful approach for detecting small changes in biologically based samples. Aromatic residues were found to contribute to peptide gel conductivity, with the importance of the peptide confirmation and fibril assembly demonstrated both experimentally and computationally. This is a small step (in combination with parallel research under way by other researchers) toward developing effective peptide-based conducting nanowires, opening the door to the use of electronics in water and physiological environments for bioelectronic and bioenergy applications.
While the application of cryogenic electron microscopy (cryo-EM) to helical polymers in biology has a long history, due to the huge number of helical macromolecular assemblies in viruses, bacteria, archaea, and eukaryotes, the use of cryo-EM to study synthetic soft matter noncovalent polymers has been much more limited. This has mainly been due to the lack of familiarity with cryo-EM in the materials science and chemistry communities, in contrast to the fact that cryo-EM was developed as a biological technique. Nevertheless, the relatively few structures of self-assembled peptide nanotubes and ribbons solved at near-atomic resolution by cryo-EM have demonstrated that cryo-EM should be the method of choice for a structural analysis of synthetic helical filaments. In addition, cryo-EM has also demonstrated that the self-assembly of soft matter polymers has enormous potential for polymorphism, something that may be obscured by techniques such as scattering and spectroscopy. These cryo-EM structures have revealed how far we currently are from being able to predict the structure of these polymers due to their chaotic self-assembly behavior.
Peptides are nature’s molecular building blocks for protein self-assembly, consisting of a sequence of amino acids covalently bound through a peptide bond. By varying the quantity, position, and type of amino acids in the peptide sequence, peptides can be tailored to deliver various chemical and physical properties. Through such tailoring, peptides become useful candidates for material self-assembly in a variety of applications, such as biotechnology, drug delivery, and bioelectronics. Typically, peptides are considered as insulators (i.e., not electrically conductive); however, microbial nanowires, composed entirely of protein, in bacteria such as G. sulfurreducens have impressive electrical properties. We have designed peptides inspired by characteristic sequences of these microbial nanowires to make conductive peptide structures. This will help us to better understand the underlying mechanisms of electron transfer in complex biological structures and understand the parameters that contribute to and promote electrical conductivity in peptides.Based on the literature, the presence of aromatic residues and the physical structure and configuration of the peptides within that structure play an important role in conferring conductivity to microbial nanowires. Therefore, we designed a 21-mer negative control peptide design (without any aromatic residues), labelled L6, with a predominantly α-helical structure that self-assembles into a fibrillar structure. To introduce electrical conductivity, the aliphatic residues in the hydrophobic core of the L6 coiled coil structure were substituted with different types and combinations of selected aromatic residues (phenylalanine (Phe) and/or tryptophan (Trp)). The introduction of aromatic residues disturbed a portion of the peptide’s α-helical structure: the design containing six Trp residues, labelled W6, showed the lowest α-helical structure and resulted in non-fibrillar assemblies, while peptides containing Phe residues resulted in fibrillar assemblies. F4 and F6 (with four and six Phe residues, respectively) retained more of the α-helical structure compared to the Trp containing peptides, F4W2 and W6 (with four and six Trp residues, respectively). Current-voltage-time (I/V-t) measurements were used to measure the intrinsic conductivity of the peptide by applying a DC voltage for 100 s over a dried peptide film deposited on interdigitated microelectrode arrays with 5 μm gaps. All aromatic containing designs showed improved conductivity over the L6 control. W6 showed the highest improvement in conductivity, followed by F4W2, indicating that including Trp in the peptide design improves the peptide electrical conductivity more than Phe. This result is in agreement with the literature, reporting that Trp amino acids have more efficient electron transfer properties thanPhe amino acids. A longer peptide was designed to stabilise the secondary structure, called W4-29mer, increasing the peptide length to 29-mer. Adding another heptad repeat (based on the L6 design) and placing four Trp residues in the second and third heptad repeats resulted in a fibril-forming peptide with an α-helical structure similar to L6, overcoming the destabilising effect of Trp. The conductivity of the W4-29mer was between that of F4W2 and W6, improving conductivity while retaining the α-helical structure. However, conductivity values were insignificant (nS.cm-1 scale) compared to the highest conductive peptide reported in the literature, ACC-Hex (mS.cm-1 range). To compare the results with the literature, the 29-mer ACC-Hex peptide was tested under the same conditions used for the peptides designed here. In addition, to improve the ACC-Hex conductivity further, a new peptide was designed (ACC-HexW) with all four Phe residues in ACC-Hex substituted with Trp residues. The I/V-t results for both peptides were found to be similar to F6 and F4. Applying the same experimental procedure for assemblies of ACC-Hex in the literature still resulted in similar conductivity values, highlighting the technical challenges of replicating data in this field. Hence, a contactless method was trialled as a proof-of-concept to measure assemblies’ conductivity by terahertz (THz) spectroscopy. W6 and L6 showed a similar trend using THz spectroscopy compared to I/V-t measurement, where W6 found to be more conductive than L6.In addition to the design parameters of the peptide, the effect of external conditions such as environmental conditions and the addition of other chemical compounds are crucial to designing functional bioelectronic materials. Increasing humidity levels increased peptide conductivity while measuring the I/V-t of all 21-mer designs. This was attributed to the ability of charged polar residues to contribute to proton conduction. Trp-containing peptides showed a higher increase in conductivity relative to humidity levels compared to other peptides, likely due to the hydrogen bonding ability of the amide group in the Trp indole ring contributing to proton conduction. Addition of chemical compounds expected to alter the electron-carrying capacity of the assemblies, such as pyrene and carbon nanotubes (CNTs), were investigated on F4W2 and W6 using the established I/V-t method. While the addition of pyrene, an aromatic compound, did not improve the conductivity of F4W2 and W6, the addition of CNT improved peptide conductivity. Additionally, the peptides dispersed and debundled the CNTs which may be promising for using these peptides in composite bioelectronic and biosensor applications.
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