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Molecular crowding plays a significant role in regulating molecular conformation in cellular environments. It is also likely to be important wherever high molecular densities are required, for example in surface-phase studies, in which molecular densities generally far exceed those observed in solution. Using on-surface circular dichroism (CD) spectroscopy, we have investigated the structure of a synthetic peptide assembled into a highly packed monolayer. The immobilized peptide undergoes a structural transition between α-helical and random coil conformation upon changes in pH and ionic concentration, but critically the threshold for conformational change is altered dramatically by molecular crowding within the peptide monolayer. This study highlights the often overlooked role molecular crowding plays in regulating molecular structure and function in surface-phase studies of biological molecules., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Numerous nanoscale devices and materials have been fabricated in recent years using a variety of biological scaffolds. However, the interfacing of these devices and materials into existing circuits and ordered arrays has proved problematic. Here, we describe a simple solution to this problem using self-assembly of the peptide coiled-coil heterodimer ACID:BASE to immobilize M13 bacteriophage particles to specific locations on a patterned gold surface. Surface plasmon resonance demonstrated that free ACID peptides will assemble onto a surface derivatized with BASE. We then displayed the ACID peptide on the pIX coat protein of M13 and showed that these phage particles permit formation of the coiled-coil resulting in specific surface attachment. The ACID:immobilized BASE affinities appear to be similar for free peptide and phage-displayed ACID. Finally, we fabricated two gold electrodes, separated by a 200 nm gap, coated one of them with BASE and showed that this allows localization of the M13:ACID onto the functionalized electrode.

The coiled coil is a widespread protein motif responsible for directing the assembly of a wide range of protein complexes. To date, research has focused largely on the solution phase assembly of coiled-coil complexes. Here, we describe an investigation into coiled-coil heterodimer assembly where one of the peptides is immobilized directly onto a gold electrode. Immobilization is achieved by the introduction of a unique cysteine residue at the C terminus, allowing for covalent and orientated attachment to a thiol-reactive surface, here the gold electrode. We show an electrochemical impedance of the resulting self-assembled polypeptide monolayer around |Z| = 4 × 10(4) Ω cm(2) at 100 mHz with a minimum phase angle of -84°, consistent with the formation of a densely packed, insulating layer. The thickness of the peptide monolayer, as measured using ellipsometry, is around 3 nm, close to that expected for a self-assembled monolayer assembled from helical peptides. Crucially, we find that the efficiency of dimerization between a peptide in solution and its coiled-coil partner peptide immobilized on a surface is strongly dependent upon the density of the immobilized peptide layer, with dimer assembly being strongly suppressed by high-density peptide monolayers. We thus develop an approach for controlling the density of the immobilized peptide by diluting the monolayer with a thiolated, random-coil peptide to modulate dimerization efficiency and demonstrate electrochemical detection of highly specific, coiled-coil heterodimer on-surface assembly.

The development of high sensitivity biosensors, for example for clinical diagnostics, requires the identification of suitable receptor molecules which offer high stability, specificity and affinity, even when embedded into solid-state biosensor transducers. Here, we present an electrochemical biosensor employing small synthetic receptor proteins (Mw ＜ 15 kDa) which emulate antibodies but with improved stability, sensitivity and molecular recognition properties, in particular when immobilized on a solid sensor surface. The synthetic receptor protein is a non-antibody-based protein scaffold with variable peptide regions inserted to provide the specific binding, and was designed to bind anti-myc tag antibody (Mw∼ 150 kDa), as a proof-of-principle exemplar. Both the scaffold and the selected receptor protein were found to have high thermostability with melting temperatures of 101 °C and 85 °C, respectively. Furthermore, the secondary structures of the receptor protein were found to be very similar to that of the original native scaffold, despite the insertion of variable peptide loops that create the binding sites. A label-free electrochemical sensor was fabricated by functionalising a microfabricated gold electrode with the receptor protein. A change in the phase of the electrochemical impedance was observed when the biosensor was subjected to anti-myc tag antibodies at concentrations between 6.7 pM and 6.7 nM. These findings demonstrate that these non-antibody receptor proteins are excellent candidates for recognition molecules in label-free biosensors. [ABSTRACT FROM AUTHOR]