Selective Detection of Protein Secondary Structural Changes in Solution Protein−Polysaccharide Complexes Using Vibrational Circular Dichroism (VCD)

A major challenge to understanding the fundamental structural basis of interactions between macromolecules in solution is how to measure their separate contributions. Particularly challenging is the interaction between proteins and polysaccharides. The polysaccharide component is often both very large (10 kDa−MDa) and immobile, or it undergoes anisotropic motion in solution, causing line broadening in NMR. Furthermore, they often exhibit signals in the very spectral regions normally employed for protein secondary structural analysis (FTIR and CD), and these signals cannot simply be subtracted because they occupy variable positions. The selective detection of protein secondary structural changes in aqueous complexes of proteins and polysaccharides, particularly the biologically important glycosaminoglycan class, is demonstrated here, exploiting a property of vibrational circular dichroism (VCD) that allows signals from proteins to be selectively detected. We show that polysaccharides, in contrast to proteins, which show well-documented and characteristic VCD signals for distinct secondary structural types, exhibit no VCD signals in the amide I‘ region despite containing N-acetyl groups. This is because the chromophores in the polysaccharides (in the CO bonds of N-acetyl and carboxylic acids groups) lack the regular geometric relationship to each other that characterizes stretches of defined protein secondary structure. We have exploited this hitherto unreported feature of VCD to enhance the contrast between proteins and bound polysaccharides in protein−polysaccharide complexes in solution. This enables the direct observation of protein secondary structural changes in protein−polysaccharide complexes in solution and will advance understanding of the structural basis of these interactions

The Impact of <i>E</i>−<i>Z</i> Photo-Isomerization on Single Molecular Conductance

The single molecule conductance of the E and Z isomers of 4,4′-(ethene-1,2-diyl)dibenzoic acid has been determined using two scanning tunneling microscopy (STM) methods for forming molecular break junctions [the I(s) (I = current and s is distance) method and the in situ break junction technique]. Isomerization leads to significant changes in the electrical conductance of these molecules, with the Z isomer exhibiting a higher conductance than the E isomer. Isomerization is achieved directly on the gold surface through photoirradiation, and the STM is used to determine conductance before and after irradiation; reversible switching between the two isomers could be achieved through irradiation of the surface bound species at different wavelengths. In addition, three groups of molecular conductance values [A (“low”), B (“medium”), and C (“high”)] have been measured for these carboxylate-terminated molecules. The origin of these conductance groups as well as the increase of the conductance for the Z isomer have been analyzed by comparing the length of the molecules extended in the gap, derived from molecular modeling, with the experimentally observed break-off distance for both isomers

Electrochemical Scanning Tunneling Spectroscopy of Redox-Active Molecules Bound by Au−C Bonds

Electrochemical Scanning Tunneling Spectroscopy of Redox-Active Molecules Bound by Au−C Bond

Substrate Structural Effects on the Synthesis and Electrochemical Properties of Platinum Nanoparticles on Highly Oriented Pyrolytic Graphite

Platinum nanoparticles have been prepared by potentiostatic multipulse electrodeposition with controlled nucleation and growth on freshly cleaved and electrochemically oxidized highly oriented pyrolytic graphite. The influence of the applied potential sequence on the size distribution was investigated. For short electrolysis times, the deposition of nanoparticles takes place via a progressive nucleation mechanism. A narrow size distribution was obtained by controlling independently the nucleation and growth steps, and particles with heights between 52 and 1.4 nm could be prepared by altering the pulse parameters. Anodic oxidation of the substrate had a large influence on the particle size, resulting in the preparation of particles 1.4 nm in height. XPS demonstrated that Pt particles of small size were readily oxidized. The rate of electrochemical methanol oxidation showed a dependence on the particle size, and no oxidation of methanol could be observed for the smaller sizes investigated

Influence of Conformational Flexibility on Single-Molecule Conductance in Nano-Electrical Junctions

The temperature dependence of the single-molecule conductance of conformationally flexible alkanedithiol molecular bridges is compared to that of more rigid analogues which contain cyclohexane ring(s). Molecular conductance has been measured with a scanning tunneling microscope (STM) at fixed gap separation by observing the stochastic formation of molecule bridges between a gold STM tip and substrate (the so-called “I(t)” technique). Under these conditions, the junction can be populated by a wide distribution of conformers of alkanedithiol molecular bridges and a strong temperature dependence of the single-molecule conductance is observed. By contrast the rigid analogues that contain cyclohexane ring(s), which cannot form the thermally accessible gauche rich conformers open to the alkanedithiols, show no dependence of the single-molecule conductance on temperature. This comparison demonstrates that it is the conformational flexibility and access to thermally populated higher energy conformers of the linear polymethylene (alkane) bridges which leads to the temperature dependence. By removing this possibility in the cyclohexane ring-containing bridges, this conformational gating is excluded and the temperature dependence is then effectively suppressed

Effect of Molecular Structure on Electrochemical Phase Behavior of Phospholipid Bilayers on Au(111)

Lipid bilayers form the basis of biological cell membranes, selective and responsive barriers vital to the function of the cell. The structure and function of the bilayer are controlled by interactions between the constituent molecules and so vary with the composition of the membrane. These interactions also influence how a membrane behaves in the presence of electric fields they frequently experience in nature. In this study, we characterize the electrochemical phase behavior of dipalmitoylphosphatidylcholine (DPPC), a glycerophospholipid prevalent in nature and often used in model systems and healthcare applications. DPPC bilayers were formed on Au(111) electrodes using Langmuir–Blodgett and Langmuir–Schaefer deposition and studied with electrochemical methods, atomic force microscopy (AFM) and in situ polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). The coverage of the substrate determined with AFM is in accord with that estimated from differential capacitance measurements, and the bilayer thickness is slightly higher than for bilayers of the similar but shorter-chained lipid, dimyristoylphosphatidylcholine (DMPC). DPPC bilayers exhibit similar electrochemical response to DMPC bilayers, but the organization of molecules differs, particularly at negative charge densities. Infrared spectra show that DPPC chains tilt as the charge density on the metal is increased in the negative direction, but, unlike in DMPC, the chains then return to their original tilt angle at the most negative potentials. The onset of the increase in the chain tilt angle coincides with a decrease in solvation around the ester carbonyl groups, and the conformation around the acyl chain linkage differs from that in DMPC. We interpret the differences in behavior between bilayers formed from these structurally similar lipids in terms of stronger dispersion forces between DPPC chains and conclude that relatively subtle changes in molecular structure may have a significant impact on a membrane’s response to its environment

Ionic Liquid Based Approach for Single-Molecule Electronics with Cobalt Contacts

An electrochemical method is presented for fabricating cobalt thin films for single-molecule electrical transport measurements. These films are electroplated in an aqueous electrolyte, but the crucial stages of electrochemical reduction to remove surface oxide and adsorption of alkane­(di)­thiol target molecules under electrochemical control to form self-assembled monolayers which protect the oxide-free cobalt surface are carried out in an ionic liquid. This approach yields monolayers on Co that are of comparable quality to those formed on Au by standard self-assembly protocols, as assessed by electrochemical methods and surface infrared spectroscopy. Using an adapted scanning tunneling microscopy (STM) method, we have determined the single-molecule conductance of cobalt/1,8-octanedithiol/cobalt junctions by employing a monolayer on cobalt and a cobalt STM tip in an ionic liquid environment and have compared the results with those of experiments using gold electrodes as a control. These cobalt substrates could therefore have future application in organic spintronic devices such as magnetic tunnel junctions

New Insights into Single-Molecule Junctions Using a Robust, Unsupervised Approach to Data Collection and Analysis

We have applied a new, robust and unsupervised approach to data collection, sorting and analysis that provides fresh insights into the nature of single-molecule junctions. Automation of tunneling current-distance (<i>I</i>(<i>s</i>)) spectroscopy facilitates the collection of very large data sets (up to 100 000 traces for a single experiment), enabling comprehensive statistical interrogations with respect to underlying tunneling characteristics, noise and junction formation probability (JFP). We frequently observe unusual low-to-high through-molecule conductance features with increasing electrode separation, in addition to numerous other “plateau” shapes, which may be related to changes in interfacial or molecular bridge structure. Furthermore, for the first time we use the JFP to characterize the homogeneity of functionalized surfaces at the nanoscale

Single-Molecule Junction Formation in Deep Eutectic Solvents with Highly Effective Gate Coupling

The environment surrounding a molecular junction affects its charge-transport properties and, therefore, must be chosen with care. In the case of measurements in liquid media, the solvent must provide good solvation, grant junction stability, and, in the case of electrolyte gating experiments, allow efficient electrical coupling to the gate electrodes through control of the electrical double layer. We evaluated in this study the deep eutectic solvent mixture (DES) ethaline, which is a mixture of choline chloride and ethylene glycol (1:2), for single-molecule junction fabrication with break-junction techniques. In ethaline, we were able to (i) measure challenging and poorly soluble molecular wires, exploiting the improved solvation capabilities offered by DESs, and (ii) efficiently apply an electrostatic gate able to modulate the conductance of the junction by approximately an order of magnitude within a ∼1 V potential window. The electrochemical gating results on a Au–VDP–Au junction follow exceptionally well the single-level modeling with strong gate coupling (where VDP is 1,2-di­(pyridine-4-yl)­ethene). Ethaline is also an ideal solvent for the measurement of very short molecular junctions, as it grants a greatly reduced snapback distance of the metallic electrodes upon point-contact rupture. Our work demonstrates that DESs are viable alternatives to often relatively expensive ionic liquids, offering good versatility for single-molecule electrical measurements

Single-Molecule Electrochemical Gating in Ionic Liquids

The single-molecular conductance of a redox active molecular bridge has been studied in an electrochemical single-molecule transistor configuration in a room-temperature ionic liquid (RTIL). The redox active pyrrolo-tetrathiafulvalene (pTTF) moiety was attached to gold contacts at both ends through −(CH₂)₆S– groups, and gating of the redox state was achieved with the electrochemical potential. The water-free, room-temperature, ionic liquid environment enabled both the monocationic and the previously inaccessible dicationic redox states of the pTTF moiety to be studied in the in situ scanning tunneling microscopy (STM) molecular break junction configuration. As the electrode potential is swept to positive potentials through both redox transitions, an ideal switching behavior is observed in which the conductance increases and then decreases as the first redox wave is passed, and then increases and decreases again as the second redox process is passed. This is described as an “off–on–off–on–off” conductance switching behavior. This molecular conductance vs electrochemical potential relation could be modeled well as a sequential two-step charge transfer process with full or partial vibrational relaxation. Using this view, reorganization energies of ∼1.2 eV have been estimated for both the first and second redox transitions for the pTTF bridge in the 1-butyl-3-methylimidazolium trifluoromethanesulfonate (BMIOTf) ionic liquid environment. By contrast, in aqueous environments, a much smaller reorganization energy of ∼0.4 eV has been obtained for the same molecular bridge. These differences are attributed to the large, outer-sphere reorganization energy for charge transfer across the molecular junction in the RTIL