Photoswitchable peptide-based ‘on-off’ biosensor for electrochemical detection and control of protein-protein interactions

Neuronal nitric oxide synthase (nNOS) is an enzyme responsible for catalyzing the production of the crucial cellular signalling molecule, nitric oxide (NO), through its interaction with the PDZ domain of α-syntrophin protein. In this study, a novel light-driven photoswitchable peptide-based biosensor, modelled on the nNOS β-finger, is used to detect and control its interaction with α-syntrophin. An azobenzene photoswitch incorporated into the peptide backbone allows reversible switching between a trans photostationary state devoid of secondary structure, and a cis photostationary state possessing a well-defined antiparallel β-strand geometry, as revealed by molecular modelling. Electrochemical impedance spectroscopy (EIS) is used to successfully detect the interaction between the gold electrode bound peptide in its cis photostationary state and a wide range of concentrations of α-syntrophin protein, highlighting both the qualitative and quantitative properties of the sensor. Furthermore, EIS demonstrates that the probe in its random trans photostationary state does not bind to the target protein. The effectiveness of the biosensor is further endorsed by the high thermal stability of the photostationary state of the cis-isomer, and the ability to actively control biomolecular interactions using light. This approach allows detection and control of binding to yield a regenerable on-off biosensor.John R.Horsley, Jingxian Yu, Kate L.Wegener, Christian Hoppmann, Karola Rück-Braun, Andrew D.Abel

Hemithioindigo-based photoswitches as ultrafast light trigger in chromopeptides

The spectroscopy and dynamics of a novel hemithioindigo-based photoswitch forming a ω-amino acid derivative are presented. Light absorption in the visible spectral range induces photoisomerization between Z and E configurations with quantum yields in the 10% range. The Z isomer is thermally stable, while the E isomer relaxes back to the Z form within several hours. The E isomers provides a distinct spectral range, where the photoisomerization process can be initiated selectively. Both directions of the photoisomerization are investigated by transient absorption spectroscopy and time constants for the formation of the photoproduct in the 10–30 ps range are observed. The ability of the hemithioindigo-based photoswitch to drive structural dynamics in peptides and proteins is tested for two ω-amino acid derivatives forming linear and cyclic structures.

Photochromic Bis(thiophen-3-yl)maleimides Studied with Time-Resolved Spectroscopy

The dynamics of the ring-closure reaction of three different bis(thiophen-3-yl)maleimides are investigated using ultrafast spectroscopy in the visible range. The structures of the molecules differ with respect to substitution of the thiophene ring and the maleimide. The experiments reveal reaction kinetics which point to the population of an excited electronic state for several nanoseconds. In the case of completely unsubstituted thiophene rings, a long excited-state lifetime (biexponential decay with 3 and 15 ns) can be observed. The remaining ultrafast absorption transients of this molecule are due to relaxational processes on the excited electronic potential energy surface. The ring-closure reaction has a small yield (<1%) and does not show up in the ultrafast absorption experiments. A dimethyl substitution of the thiophene ring results in completely different behavior: after transients related to relaxation in the excited electronic state, one finds pronounced absorption transients with τ = 16 ps which represent the partial decay of the excited electronic state and the formation of the ring-closed isomer. Another fraction of the emitting excited electronic state decays again on the few nanosecond time scale. The experiments suggest that the open isomer of the dimethyl-substituted imides exists in two conformations.

Light-Switchable Hemithioindigo−Hemistilbene-Containing Peptides: Ultrafast Spectroscopy of the Z → E Isomerization of the Chromophore and the Structural Dynamics of the Peptide Moiety

Two hemithioindigo−hemistilbene (HTI) derivatives, designed to operate as structural switches in peptides, as well as two HTI peptides are characterized by ultrafast spectroscopy in the visible and the infrared. The two HTI switches follow the reaction scheme published for other HTI compounds with a picosecond excited state reaction (τ1 ≈ 6 ps) and isomerization from Z to E with τ2 = 13 and 51 ps. As compared to the isolated chromophores, the isomerization reaction is slowed down in the chromopeptides to τ2 = 24 and 69 ps. For the smaller peptide containing 6 amino acids, the structural changes of the peptide moiety observed via the IR spectrum in the amide I band follow the isomerization of the molecular switch closely. In the larger cyclic chromopeptide, containing 20 amino acids and mimicking a β-hairpin structure in the Z-form of the chromophore, the peptide moiety also changes its structure during isomerization of the chromophore. However, the IR spectrum at the end of the observation period of 3 ns deviates significantly from the stationary difference spectrum. These signatures indicate that strong additional structural changes, e.g., breaking of interchain hydrogen bonds, also occur on longer time scales.