Christian Hoppmann

Light‐Directed Protein Binding of a Biologically Relevant β‐Sheet

b-Hairpin structures are frequently involved in protein– protein interactions that control essential processes in cells and are therefore interesting targets for interference. Hairpinforming peptides that compete with such protein interactions are valuable tools for studying biological processes. Moreover, the incorporation of a photoswitchable unit into appropriate b-hairpin-forming peptide ligands could allow protein interactions in cells to be studied by light-triggered interference. However, b-hairpin structures are rarely studied because of the limited availability and stability of suitable model peptides. This is because such a model peptide has to fulfill at least three requirements: 1) the b-hairpin has to be sufficiently stable as monomer without the tendency to selfaggregate, 2) the photoswitchable unit incorporated must stabilize the biologically active peptide conformation, and 3) disturbing the protein binding site by light-induced isomerization of the photoswitch must not result in intermolecular association or even formation of insoluble fibrils. Herein, we report the first example of a b-hairpin model peptide of a biologically important protein domain that shows considerably different binding affinities for the target protein that are dependent on the isomerization state of the embedded photoswitch. PDZ domains mediate the formation of a variety of multiprotein complexes in the cell. Besides C-terminal protein sequences, PDZ domains are also able to recognize internal peptide motifs that bind at the same binding pocket as the C-terminal ones. The best example of this type of internal ligand recognition is found in the extended PDZ domain of neuronal nitric oxide synthase (nNOS) which interacts with the PDZ domain from a-1-syntrophin or the second PDZ domain from PSD95. The formation of the PDZ/PDZ heterodimer requires the b-finger structure of nNOS (30 amino acid residues) to bind at the syntrophin PDZ domain, thus mediating the membrane association of nNOS to skeletal muscle and inducing the production of the second messenger nitric oxide (NO) for muscle contraction. Crucial for binding is the internal recognition motif -LETTFof the extended PDZ domain of nNOS located in the first strand of the hairpin peptide (Scheme 1), a stable

Photocontrol of Contracting Muscle Fibers

The light-controlled inhibition of physiologically relevant protein–protein interactions by appropriate photoresponsive ligands in living cells or small organs (skeletal muscle fibers, vessels) could make it possible to investigate signaling pathways under high spatiotemporal control. Recently, we have reported a cyclic peptide that mimics the b-finger motif in neural NO synthase (nNOS) which is crucial for binding of nNOS to a-1-syntrophin. When a photoswitchable unit is embedded into this peptide, the binding can be controlled in vitro simply by light. In skeletal muscle the extended PDZ domain of nNOS interacts with the PDZ domain of a-1syntrophin to recruit nNOS to the dystrophin-associated protein complex in the plasma membrane, thus coupling the production of the second messenger nitric oxide (NO) to muscle contraction. Loss of sarcolemmal nNOS is known to result in functional ischemia during muscle contraction, which is commonly observed in muscle diseases as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Herein we show that the photoswitchable peptide ligand 1 (Figure 1) is able to translocate into cells, is sufficiently stable towards intracellular conditions, and can be used in vivo to photocontrol contracting muscle fibers. The recently described nNOS-derived, photoswitchable peptide ligand 1 of a-1-syntrophin contains the azobenzenew-amino acid 3-((4’-aminomethyl)phenylazo)benzoic acid (3,4’-AMPB), which in its trans form led to a ligand that showed no affinity to the PDZ domain of a-1-syntrophin while photoisomerization to the cis form resulted in a remarkable affinity of the peptide (KD = 10.6 mm). [1a] The finding has addressed the question of the applicability of the light-controlled ligand under physiological conditions to investigate the native interference in living skeletal muscles. Light-directed binding of the cis form of the photoswitchable ligand 1 to syntrophin was expected to inhibit the native syntrophin–nNOS interaction in the skeletal muscle followed by the dislocation of nNOS from the sarcolemma which may result in reduced NO release from skeletal muscle cells and thus in light-controlled muscle contraction. Azobenzene units have been used extensively for the photomodulation of biomolecules (peptides, proteins, and nucleic acids) and biological processes in vitro and in vivo (as in ion channels). The feasibility of using azobenzene systems in living organisms to photocontrol biological events has been confirmed by the in vivo imaging of the isomerization process in zebrafish. An intrinsic hindrance for applications of azobenzene in living cells derives from its susceptibility to reduction. The azo unit may be subject to reduction by enzymes or thiols such as glutathione (GSH) which is present in most cells at millimolar concentration (0.5–10 mm). The reduction rate of the cis isomer of a parasubstituted AMPB amino acid in a model tripeptide is about 100-fold higher than for the corresponding trans isomer. To determine the stability of the AMPB switch unit in the peptide ligand 1 we incubated the cis form of the photoswitchable ligand at the photostationary state (pss) in buffer solution (pH 7.5) containing reduced glutathione (10 mm). After 1 h exposure to GSH no reduced material was detectable by LC–MS analysis (Figure 2, dashed line). Even after 16 h the reduced material amounted to only 5% (Figure 2, dotted line, signal marked with an arrow; Figure S3 in the Supporting Information). In addition, when changes in the UV/Vis spectra of the photoswitchable ligand were followed during irradiation in buffer solution (pH 7.5) containing GSH (10 mm), isosbestic points were retained, indicating the stability of the 3,4’-AMPB unit in the peptide ligand 1 (Figure S1 in the Supporting Information). As expected, the thermal cis!trans isomerization of the photoswitchable ligand 1 in the presence of 10 mm glutathione was Figure 1. Structure of the cis form of the photoswitchable peptide ligand related to the b-finger peptide of nNOS.

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 (τ(1) ≈ 6 ps) and isomerization from Z to E with τ(2) = 13 and 51 ps. As compared to the isolated chromophores, the isomerization reaction is slowed down in the chromopeptides to τ(2) = 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.

Photoswitchable click amino acids: light control of conformation and bioactivity.

Click the switch: By using a photoswitchable click amino acid (PSCaa) a light-induced intramolecular thiol-ene click reaction with a neighboring cysteine under very mild conditions results in an azobenzene bridge. By expanding the genetic code for PSCaa the specific incorporation of photoswitch units into proteins in living cells can result in an exciting approach for studying light-controllable activity, in vivo.

Intramolecular bridges formed by photoswitchable click amino acids.

Summary Photoswitchable click amino acids (PSCaa) are amino acids bearing a side chain consisting of a photoswitchable unit elongated with a functional group that allows for a specific click reaction, such as an alkene that can react with the thiol group of a cysteine residue. An intramolecular click reaction results in the formation of a photoswitchable bridge, which can be used for controlling conformational domains in peptides and proteins. The ability to control conformations as well as the efficiency of the intramolecular bridging depends on the length of the PSCaa side chain and the distance to the cysteine residue to be clicked with. On comparing i,i+4 and i,i+7 spacings of PSCaa and cysteine in a model peptide without a preferred conformation, it was seen that the thiol–ene click reaction takes place efficiently in both cases. Upon induction of an α-helical structure by the addition of trifluoroethanol, the thiol click reaction occurs preferentially with the i,i+4 spacing. Even in the presence of glutathione as an additional thiol the click reaction of the PSCaa occurs intramolecularly with the cysteine rather than with the glutathione, indicating that the click reaction may be used even under reducing conditions occurring in living cells.

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.