Peptide Self-assembled Monolayers Research Articles

Deciphering the Roles of Interfacial Amino Acids in Inter-Protein Charge Transport.

Elucidating charge transport (CT) through proteins is critical for gaining insights into ubiquitous CT chain reactions in biological systems and developing high-performance bioelectronic devices. While intra-protein CT has been extensively studied, crucial knowledge about inter-protein CT via interfacial amino acids is still absent due to the structural complexity. Herein, by loading cytochrome c (Cyt c) on well-defined peptide self-assembled monolayers to mimic the protein-protein interface, we provide a precisely controlled platform for identifying the roles of interfacial amino acids in solid-state CT via peptide-Cyt c junctions. The terminal amino acid of peptides serves as a fine-tuning factor for both the interfacial interaction between peptides and Cyt c and the immobilized Cyt c orientation, resulting in a nearly 10-fold difference in current through peptide-Cyt c junctions with varied asymmetry. This work provides a valuable platform for studying CT across proteins and contributes to the understanding of fundamental principles governing inter-protein CT.

Tuning Charge Transport of Oligopeptide Junctions via Interfacial Amino Acids†

Comprehensive SummaryThe charge transport through peptides can imitate the corresponding processes in more complicated proteins, enabling us to develop high‐performance bioelectronic devices and to understand the mechanisms of biomolecular recognition and information transfer. While charge transport modulation through individual peptides has been achieved via various covalent strategies, the intermolecular modulation is still very challenging, which may capture the charge transport between proteins. To tackle this challenge, we used well‐defined self‐assembled monolayers (SAMs) of oligopeptides as a model to imitate the interface of proteins and explored an interfacial amino acid strategy for charge transport modulation. We showed that non‐covalently interfaced charged amino acids (e.g., arginine) effectively attenuated the charge transport of glutamic acid terminated polyglycine peptide SAMs. By analyzing the relationship of the charge transport with the molecular frontier orbital relative to the Fermi energy level of the electrode, the molecule‐electrodes coupling (Γ), and the trends in skewness and kurtosis with voltage and the dielectric constant (εr), we showed that the attenuation was from the decreased Γ and the reduced polarizability. We present an efficient strategy to modulate the charge transport of oligopeptide‐SAM junctions by intermolecular interactions, which will advance our understanding of charge transport in biological systems and facilitate developing future electronics.

Charge migration of ferrocene-labeled peptide self-assembled monolayers at various interfaces: The roles of peptide composition

Understanding charge migration across peptide chain of proteins is important for both biology and biomolecular electronics. Despite experimental and theoretical researches showing that the charge migration in wet electrochemistry and on solid state are interrelated, there are still limited insights about this interrelation. Here, taken ferrocene-labeled peptide self-assembled monolayers on the Au substrate as a model, the relation between the charge migration on solid state (charge transport, CTp) and in wet electrochemistry (charge transfer, CT) was investigated. By characterizing the current density from solid state molecular junctions and the electron transfer rate (kET) from electrochemical measurement in solution, we found that the ability of peptides to transmit charge was more sensitive to peptide's length on solid state than that in wet electrochemistry, comparing to the reported alkane and peptide nucleic acid oligomers. In addition, changing peptide compositions, such as doping phenylalanine (F) or aspartic acid (D) into the initial glycine (G) peptide backbone, made a different consequence for charge migration: the kET in wet electrochemistry was F > G ≈ D, while the CTp rate on solid state was G ≈ F > D. Our findings provide further insights into the comprehension of relations between charge migration and peptide structures.

The role of externally-modulated electrostatic interactions in amplifying charge transport across lysine-doped peptide junctions

Many evolved biomolecular functions such as ion pumping or redox catalysis rely on controlled charge transport through the polypeptide matrix, which can be regulated by shifts in molecular protonation states and dependent supramolecular packing modes in response to environmental cues. However, the exact roles of such dynamic, non-covalent interactions in peptide charge transport have remained elusive. To tackle this challenge, here we report the modulation of charge transport in a series of lysine (Lys)-substituted hepta-glycine (Gly) peptide self-assembled monolayers (SAMs) on template-striped gold (AuTS) bottom electrodes with eutectic gallium-indium (EGaIn) liquid metal top electrodes. We demonstrate systematic modulation of hydrogen bonding and more general electrostatic interactions by shifting the position of the charged Lys-residue and creating different protonation patterns by changing the environmental pH in the AuTS/peptide//GaOx/EGaIn junctions. The effective modulation is evidenced by current density–voltage (J-V) measurements combined with SAM characterization using ultraviolet photoelectron spectroscopy (UPS) and angle-resolved X-ray photoelectron spectroscopy (ARXPS), polarization modulation–infrared reflection-absorption spectroscopy (PM-IRRAS), and molecular dynamics (MD) simulations. Decreasing the hydrogen bonding inside the peptide SAMs and increasing the electrostatic interactions by environmental counterions amplifies the charge transport differently with Lys-position, which means that the sensitive electrical response of peptide SAMs can be tuned by the peptide sequence. Our results provide insights into the relationship between molecular design and in situ modulation of charge transport properties for the development of bionanoelectronics.

Efficient Enzymatic Incorporation of Dehydroalanine Based on SAMDI-Assisted Identification of Optimized Tags for OspF/SpvC.

Site-specific modification of proteins has important applications in biological research and drug development. Reactive tags such as azide, alkyne, and tetrazine have been used extensively to achieve the abovementioned goal. However, bulky side-chain "ligation scars" are often left after the labeling and may hinder the biological application of such engineered protein products. Conjugation chemistry via dehydroalanine (Dha) may provide an opportunity for "traceless" ligation because the activated alkene moiety on Dha can then serve as an electrophile to react with radicalophile, thiol/amine nucleophile, and reactive phosphine probe to introduce a minimal linker in the protein post-translational modifications. In this report, we present a mild and highly efficient enzymatic approach to incorporate Dha with phosphothreonine/serine lyases, OspF and SpvC. These lyases originally catalyze an irreversible elimination reaction that converts a doubly phosphorylated substrate with phosphothreonine (pT) or phosphoserine (pS) to dehydrobutyrine (Dhb) or Dha. To generate a simple monophosphorylated tag for these lyases, we conducted a systematic approach to profile the substrate specificity of OspF and SpvC using peptide arrays and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. The optimized tag, [F/Y/W]-pT/pS-[F/Y/W] (where [F/Y/W] indicates an aromatic residue), results in a ∼10-fold enhancement of the overall peptide labeling efficiency via Dha chemistry and enables the first demonstration of protein labeling as well as live cell labeling with a minimal ligation linker via enzyme-mediated incorporation of Dha.

Mineralization of calcium phosphate controlled by biomimetic self-assembled peptide monolayers via surface electrostatic potentials

The functions of acidic-rich domains in non-collagenous protein during biomineralization are thought to induce nucleation and control the growth of hydroxyapatite. The tripeptide Asp-Ser-Ser (DSS) repeats are the most common acidic-rich repeated unit in non-collagenous protein of dentin phosphoprotein, the functions of which have aroused extensive interests. In this study, biomimetic peptides (DSS)n (n = 2 or 3) were designed and fabricated into self-assembled monolayers (SAMs) on Au (111) surface as biomimetic organic templates to regulate hydroxyapatite (HAp) mineralization in 1.5 simulated body fluid (1.5 SBF) at 37 °C. The early mineralization processes and minerals deposited on the SAMs were characterized by X-ray diffraction, scanning electron microscope, and Fourier transform infrared spectroscopy analyses. The SAM-DSS9/DSS9G showed the highest capacity to induce HAp nucleation and growth, followed by SAM-DSS6/DSS6G, SAM-COOH, and SAM-OH. The SAM-(DSS)n had more negative zeta potentials than SAM-COOH surface, indicating that DSS repeats contributed to the biomineralization, which not only provided strong affinity with Ca2+ ions through direct electrostatic bonds, but more importantly influence surface electrostatic potentials of the assembled organic template for nucleation.

α-Aminoisobutyric Acid-Stabilized Peptide SAMs with Low Nonspecific Protein Adsorption and Resistance against Marine Biofouling

A series of low fouling peptide self-assembled monolayers (SAMs) was developed to understand how the effects of subtle sequence alterations determine the properties of peptide-terminated SAMs and s...

Supramolecular Presentation of Hyaluronan onto Model Surfaces for Studying the Behavior of Cancer Stem Cells.

The supramolecular presentation of extracellular matrix components on surfaces provides a platform for the investigation and control of cell behavior. Hyaluronan (HA) is one of the main components of the extracellular environment and has been shown to play an important role in different cancers and their progression. However, current methods of HA immobilization often require its chemical modification. Herein, a peptide-based self-assembled monolayer (SAM) is used as an anchor to immobilize unmodified HA on a bare gold surface, as demonstrated by the quartz crystal microbalance with dissipation monitoring. Peptide-HA surfaces show increased roughness and greater hydrophobicity when compared to poly-D-lysine/HA surfaces, as measured by atomic force microscopy and water contact angle, respectively. Additionally, the peptide SAM can be micro-contact printed and used to restrict the presentation of HA to specific regions, thereby creating HA patterned surfaces to examine cell behavior. When used for cell culture, these surfaces result in altered adhesion and migration of LUC4 head and neck squamous cell carcinoma cells. These biomimetic surfaces can provide insights into the role of HA in cancer and other diseases and be used as a platform for the development of cell sorting devices.

Using Peptide Arrays To Discover the Sequence-Specific Acetylation of the Histidine-Tyrosine Dyad.

Reactions that can selectively modify amino acid sequences within peptides and proteins are important for preparing protein reagents, immobilizing proteins, and making antibody-drug conjugates. The development of new reactions often begins with known chemistries and optimizes yields using a small set of peptide reactants. This article describes the use of peptide arrays and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to discover and characterize unanticipated sequence-selective reactions of peptides. This work reports the selective acetylation of HY (histidine-tyrosine) and YH (tyrosine-histidine) dyads when treated with acetic anhydride in aqueous conditions. More broadly, this example illustrates the benefits of using peptide arrays and a label-free analysis method to discover peptide-modifying reactions and gain mechanistic insight into their sequence specificity.

Metal Binding to Aβ Peptides Inhibits Interaction with Cytochrome c: Insights from Abiological Constructs.

Aβ(1–40) peptide is mutated to introduce cysteine residue to allow formation of organized self-assembled monolayers (SAMs) on Au electrodes. Three mutants of this peptide are produced, which vary in the position of the inserted cysteine residue. Fourier transform infrared data on these peptide SAMs show the presence of both α helices and β sheet in these Aβ constructs. These peptide constructs interact with cytochrome c (Cytc), allowing electron transfer between Cytc and the electrode via the Aβ peptides. Binding of metals like Zn2+ or Cu2+ induces changes in the morphologies of these assemblies, making them fold, which inhibits their spontaneous interaction with Cytc.

Cytochrome c electrochemistry on peptide self-assembled monolayers

A short chain peptide is covalently attached to an evaporated gold electrode to form a biocompatible self-assembled monolayer (SAM) surface. The peptide is designed to optimize the interfacial interaction of horse heart cytochrome c (cytc) through the electrostatic interaction of the carboxylate group on the terminal amino acid glutamate and the amine on lysine in cytc. Cytc exhibits quasi-reversible voltammetry and is stable on the time scale of the experiments. Marcus theory is used to calculate the electron transfer rates, which are ~5 times faster for cytc/peptide/Au compared to cytc/11-mercaptoundecanoic acid/Au SAMs of similar chain length. The formal potentials also shift for the cytc/peptide system ~20 mV more positive than cytc adsorbed to 11-mercaptoundecanoic acid-SAM/Au electrodes.

Resistance of Zwitterionic Peptide Monolayers to Biofouling.

Self-assembled monolayers (SAMs) are widely used in science and engineering, and recent progress has demonstrated the utility of zwitterionic peptides with alternating lysine (K) and glutamic acid (E) residues for antifouling purposes. Aiming at developing a peptide-based fouling-resistant SAM suitable for presentation of surface-attached pheromones for barnacle larvae, we have investigated five different peptide SAMs, where four are based on the EK motif, and the fifth was designed based on general principles for fouling resistance. The SAMs were formed by self-assembly onto gold substrates via cysteine residues on the peptides, and formation of SAMs was verified via ellipsometry, wettability, infrared reflection-absorption spectroscopy and cyclic voltammetry. Settlement of cypris larvae of the barnacle Balanus (=Amphibalanus) amphitrite, the target of pheromone studies, was tested. SAMs were also subjected to fouling assays using protein solutions, blood serum, and the bacterium Mycobacterium marinum. The results confirm the favorable antifouling properties of EK-containing peptides in most of the assays, although this did not apply to the barnacle larvae settlement test, where settlement was low on only one of the peptide SAMs. The one peptide that had antifouling properties for barnacles did not contain a pheromone motif, and would not be susceptible to degredation by common serine proteases. We conclude that the otherwise broadly effective antifouling properties of EK-containing peptide SAMs is not directly applicable to barnacles, and that great care must be exercised in the design of peptide-based SAMs for presentation of barnacle-specific ligands.

Catalytic Activity of Peptide-Nanoparticle Conjugates Regulated by a Conformational Change.

Herein, we present the design and synthesis of a catalytically active peptide-nanoparticle conjugate whose activity is regulated by a defined conformational change in the self-assembled peptide monolayer. A catalytically active peptide, designed after the heterodimeric α-helical coiled-coil principle was immobilized onto gold nanoparticles, and kinetic studies were performed according to the Michaelis-Menten model. The formed peptide monolayer at the gold nanoparticle surface accelerated p-nitrophenylacetate (pNPA) hydrolysis by 1 order of magnitude compared to the soluble peptide while exhibiting no defined secondary structure as determined by infrared (IR) and circular dichroism (CD) spectroscopy. Addition of the complementary peptide-induced coiled-coil formation while significantly hindering the pNPA hydrolysis catalyzed by the peptide-nanoparticle conjugate. The heptad repeat sequence of a coiled-coil opens up the opportunity for regulation of conformation and thus catalytic activity of peptide-nanoparticle conjugates upon interaction with a complementary coiled-coil sequence. Strategies of regulation of catalytic activity by interaction with a complementary cofactor/ligand are well-established in nature and are introduced here into rationally designed peptide-nanoparticle conjugates.

Invited: Microbiology Leads the Way: From Protein Nanowires to Hybrid Devices

In Nature, highly efficient and diverse consortia of microbes cycle carbon and other elements in electrochemical reactions that discharge metabolic electrons onto soluble and insoluble metals. One group in particular, Geobacter bacteria, uses hair-like protein filaments or pili to bind and transfer electrons to iron (Fe[III]) oxides, a reaction that solubilizes part of the iron as Fe(II) and also generates the semi-conducting mineral, magnetite. Geobacter cells also use the conductive pili to bind and reduce toxic metals such as uranium (present as the uranyl divalent cation). The reaction reduces the U(VI) in the uranyl cation to a mononuclear mineral phase of U(IV), effectively precipitating the radionuclide extracellularly and preventing its permeation inside the cells. Interestingly, the Geobacter pili are composed of a single peptide subunit, which polymerizes in a helical fashion such that contacts are formed between the side chains of aromatic residues to promote fast rates of charge hopping. Structural studies also identified ligands on the pilus surface that could function as metal traps for the uranyl cation and other cationic metals. Once bound, the metals are positioned in close proximity to a terminal tyrosine residue of the pilus multistep hopping path to promote their reductive precipitation. The ability of a peptide assembly such as Geobacter pili to transport electrons at micrometer distances is especially significant for applications in nanotechnology. We show that recombinant techniques can be applied to develop sustainable manufacturing protocols for the mass-production of conductive peptides inspired in the pilus subunit or pilin. Information about the amino acids residues and structural features of the pilin that contribute to its self-assembly and conductivity was used to design protocols for their in vitro assembly in a cell-free environment, effectively mass-producing protein nanowires. Further, genetic engineering was used to functionalize the recombinant peptides with chemical tags for their selective and specific integration into nanoelectronic devices. This approach allowed us to manufacture conductive Peptide Self-Assembled Monolayers (PSAMs) on electrodes that concentrate the metal traps on the monolayer surface and maximize metal binding and reduction. The ability to custom-design and mass-produce protein nanowires and PSAMs via recombinant and genetic engineering approaches contrasts with fabrication methods for inorganic nanowires or hybrid devices with synthetic peptides, which can involve high temperatures, toxic solvents, vacuums, and/or specialized equipment, and the limited methods available for their functionalization. Protein nanowires and PSAMs also circumvent major concerns regarding cyto- and genotoxicity that limit commercial applications of carbon, metal, and metal-oxide based nanomaterials, making them suitable for the development of biodegradable and biocompatible nanoelectronic devices. Of special significance is the ability of the recombinant protein nanowires and PSAMs to retain the ability to bind and reductively precipitate cationic metal contaminants such as uranium, a process that could be harnessed to develop sensors and deployable devices for bioremediation.

Ionic Strength, Surface Charge, and Packing Density Effects on the Properties of Peptide Self-Assembled Monolayers.

The various environmental parameters of packing density, ionic strength, and solution charge were examined for their effects on the properties of the immobilized peptide mimotope CH19 (CGSGSGSQLGPYELWELSH) that binds with the therapeutic antibody Trastuzumab (Herceptin) on a gold substrate. The immobilization of CH19 onto gold was examined with a quartz crystal microbalance (QCM). The QCM data showed the presence of intermolecular interactions resulting in the increase of viscoelastic properties of the peptide self-assembled monolayer (SAM). The CH19 SAM was diluted with CS7 (CGSGSGS) to decrease the packing density as CH19/CS7. The packing density and ionic strength parameters were evaluated by atomic force microscopy (AFM), ellipsometry, and QCM. AFM and ellipsometry showed a distinct conformational difference between CH19 and CH19/CS7, indicating a relationship between packing density and conformational state of the immobilized peptide. The CH19 SAM thickness was 40 Å with a rough topology, while the CH19/CS7 SAM thickness was 20 Å with a smooth topology. The affinity studies showed that the affinity of CH19 and CH19/CS7 to Trastuzumab were both on the order of 107 M-1 in undiluted PBS buffer, while the dilution of the buffer by 1000× increased both SAMs affinities to Trastuzumab to the order of 1015 M-2 and changed the binding behavior from noncooperative to cooperative binding. This indicated that ionic strength had a more pronounced effect on binding properties of the CH19 SAM than packing density. Electrochemical impedance spectroscopy (EIS) was conducted on the CH19/CS7 SAM, which showed an increase in impedance after each EIS measurement cycle. Cyclic voltammetry on the CH19/CS7 SAM decreased impedance to near initial values. The impact of the packing density, buffer ionic strength, and local charge perturbation of the peptide SAM properties was interpreted based on the titratable sites in CH19 that could participate in the proton transfer and water equilibrium.

A label-free electrochemical DNA biosensor for breast cancer marker BRCA1 based on self-assembled antifouling peptide monolayer

Electrochemical assays potentially offer a highly cheap, automated, portable and sensitive method for a great deal of disease biomarker detections, and minimization of nonspecific adsorption is a critical issue for their application in natural complex media. We present herein, the development and application of a highly sensitive and selective electrochemical DNA hybridization biosensor for breast cancer marker BRCA1, based on a zwitterionic peptide self-assembled monolayer (SAM) support serving as the low fouling substrate, the 19-mer BRCA1 related sequence-specific oligonucleotides, and the sensitively label-free sensing technique of electrochemical impedance spectroscopy (EIS). The capability of such peptide SAM in resisting nonspecific protein adsorption was surveyed using EIS, and the biosensor fabrication process and sensing performance were also investigated with X-Ray photoelectron spectroscopy (XPS) and EIS. Significantly, this developed biosensing strategy achieved a linear detection range from 1.0fM to 10.0pM with a limit of detection of 0.3fM, associated with satisfactory sensing properties of facile fabrication, desired sensitivity, high selectivity, and favourable reproducibility and antifouling ability, manifesting its promising potential in practical application.

Dipole Moment Effect on the Electrochemical Desorption of Self‐Assembled Monolayers of 310‐Helicogenic Peptides on Gold

AbstractA series of 12 thiolated oligopeptides of α‐aminoisobutyric acid (Aib) were used to make self‐assembled monolayers (SAMs) on carefully annealed gold plates. These peptides, which form 310‐helices and have a strong dipole moment along the main peptide axis, were devised to give rise from zero to seven C=O⋅⋅⋅H−N intramolecular hydrogen bonds. The orientation of the natural dipole moment of the peptide was controlled by linking the thiolated moiety to either the nitrogen or carbon terminus. To assess the interactions between the chains forming the monolayer and determine the effect of the peptide orientation, the SAMs were characterized by polarization‐modulation infrared‐reflection absorption spectroscopy, in comparison with the FTIR behavior of the free peptides in solution. The stability of the peptide SAMs toward electroreduction was studied by cyclic voltammetry in 0.1 m NaOH. We show that electrodesorption is significantly affected by both peptide length and orientation in a way consistent with a strong effect exerted by the dipole moment of the peptide.

Superior Antifouling Performance of a Zwitterionic Peptide Compared to an Amphiphilic, Non-Ionic Peptide.

The aim of this study was to explore the influence of amphiphilic and zwitterionic structures on the resistance of protein adsorption to peptide self-assembled monolayers (SAMs) and gain insight into the associated antifouling mechanism. Two kinds of cysteine-terminated heptapeptides were studied. One peptide had alternating hydrophobic and hydrophilic residues with an amphiphilic sequence of CYSYSYS. The other peptide (CRERERE) was zwitterionic. Both peptides were covalently attached onto gold substrates via gold-thiol bond formation. Surface plasmon resonance analysis results showed that both peptide SAMs had ultralow or low protein adsorption amounts of 1.97-11.78 ng/cm2 in the presence of single proteins. The zwitterionic peptide showed relatively higher antifouling ability with single proteins and natural complex protein media. We performed molecular dynamics simulations to understand their respective antifouling behaviors. The results indicated that strong surface hydration of peptide SAMs contributes to fouling resistance by impeding interactions with proteins. Compared to the CYSYSYS peptide, more water molecules were predicted to form hydrogen-bonding interactions with the zwitterionic CRERERE peptide, which is in agreement with the antifouling test results. These findings reveal a clear relation between peptide structures and resistance to protein adsorption, facilitating the development of novel peptide-containing antifouling materials.

Anodic Photocurrent Generation by Porphyrin-Terminated Helical Peptide Monolayers on Gold

Photocurrent generation of porphyrin-terminated helical peptide self-assembled monolayers (SAMs) was studied in an aqueous solution. The anodic photocurrent was prevailing, but the cathodic photocurrent was observed with applying negative bias voltage on the working electrode. The bias dependence of the photocurrent was explained successfully by theoretical calculation with taking into account the redox potential shift by J-aggregate of porpyrins, the helix dipole, and photoenergy migration in the SAM. The dark current was insignificant even at the forward bias voltage.

Holographic microscopy provides new insights into the settlement of zoospores of the green alga Ulva linza on cationic oligopeptide surfaces

Interaction of zoospores of Ulva linza with cationic, arginine-rich oligopeptide self-assembled monolayers (SAMs) is characterized by rapid settlement. Some spores settle (ie permanently attach) in a ‘normal’ manner involving the secretion of a permanent adhesive, retraction of the flagella and cell wall formation, whilst others undergo ‘pseudosettlement’ whereby motile spores are trapped (attached) on the SAM surface without undergoing the normal metamorphosis into a settled spore. Holographic microscopy was used to record videos of swimming zoospores in the vicinity of surfaces with different cationic oligopeptide concentrations to provide time-resolved insights into processes associated with attachment of spores. The data reveal that spore attachment rate increases with increasing cationic peptide content. Accordingly, the decrease in swimming activity in the volume of seawater above the surface accelerated with increasing surface charge. Three-dimensional trajectories of individual swimming spores showed a ‘hit and stick’ motion pattern, exclusively observed for the arginine-rich peptide SAMs, whereby spores were immediately trapped upon contact with the surface.