Photoresponsive Peptide Research Articles

Photoresponsive peptide materials: Spatiotemporal control of self-assembly and biological functions.

Peptides work as both functional molecules to modulate various biological phenomena and self-assembling artificial materials. The introduction of photoresponsive units to peptides allows the spatiotemporal remote control of their structure and function upon light irradiation. This article overviews the photoresponsive peptide design, interaction with biomolecules, and applications in self-assembling materials over the last 30 years. Peptides modified with photochromic (photoisomerizable) molecules, such as azobenzene and spiropyran, reversibly photo-controlled the binding to biomolecules and nanostructure formation through self-assembly. Photocleavable molecular units irreversibly control the functions of peptides through cleavage of the main chain and deprotection by light. Photocrosslinking between peptides or between peptides and other biomolecules enhances the structural stability of peptide assemblies and complexes. These photoresponsive peptides spatiotemporally controlled the formation and dissociation of peptide assemblies, gene expressions, protein-drug interactions, protein-protein interactions, liposome deformation and motility, cytoskeleton structure and stability, and cell functions by appropriate light irradiation. These molecular systems can be applied to photo-control biological functions, molecular robots, artificial cells, and next-generation smart drug delivery materials.

Photoinduced Amyloid Fibril Degradation for Controlled Cell Patterning.

Amyloid-like fibrils are a special class of self-assembling peptides that emerge as a promising nanomaterial with rich bioactivity for applications such as cell adhesion and growth. Unlike the extracellular matrix, the intrinsically stable amyloid-like fibrils do not respond nor adapt to stimuli of their natural environment. Here, a self-assembling motif (CKFKFQF), in which a photosensitive o-nitrobenzyl linker (PCL) is inserted, is designed. This peptide (CKFK-PCL-FQF) assembles into amyloid-like fibrils comparable to the unsubstituted CKFKFQF and reveals a strong response to UV-light. After UV irradiation, the secondary structure of the fibrils, fibril morphology, and bioactivity are lost. Thus, coating surfaces with the pre-formed fibrils and exposing them to UV-light through a photomask generate well-defined areas with patterns of intact and destroyed fibrillar morphology. The unexposed, fibril-coated surface areas retain their ability to support cell adhesion in culture, in contrast to the light-exposed regions, where the cell-supportive fibril morphology is destroyed. Consequently, the photoresponsive peptide nanofibrils provide a facile and efficient way of cell patterning, exemplarily demonstrated for A549, Chinese Hamster Ovary, and Raw Dual type cells. This study introduces photoresponsive amyloid-like fibrils as adaptive functional materials to precisely arrange cells on surfaces.

Targeted antimicrobial peptide delivery in vivo to tumor with near infrared photoactivated mesoporous silica nanoparticles.

Antimicrobial peptide PA-C1b (chensinin-1b conjugated with palmitic acid) showed potent anticancer activity with no obvious hemolytic activity, which made it a potential agent for treating cancers. However, after in vivo administration, peptides can be degraded by proteases because there is no effective protection. In this study, a tumor-targeting photoresponsive antimicrobial peptide delivery system was developed, and the peptide PA-C1b labeled with the dye sulfo-cyanine7 (Cy7) was loaded into mesoporous silica nanoparticles (MSNs). The final MSN@Cy7-PA-C1b nanoparticles were wrapped by graphene oxide (GO), and then folic acid was conjugated to the surface of the MSNs for targeting purposes. The final MSN@Cy7-PA-C1b@FA-GO nanoparticles were constructed to allow light-mediated peptide release and folate receptor-targeted cancer therapy. The Cy7 dye serves as a real-time indicator, and GO acts as a gatekeeper to prevent leakage of the loaded peptides in the absence of near-infrared light irradiation. Upon light irradiation, the GO wrapping detaches, and the photoresponsive peptide delivery system works well both in in vitro cell experiments and during in vivo administration in mouse tumor experiments. The construction of the MSN@Cy7-PA-C1b@FA-GO platform provides a novel approach to deliver antimicrobial peptides in vivo for the treatment of infections by pathogenic microorganisms and cancers.

In search of visible-light photoresponsive peptide nucleic acids (PNAs) for reversible control of DNA hybridization.

Photoswitchable oligonucleotides can determine specific biological outcomes by light-induced conformational changes. In particular, artificial probes activated by visible-light irradiation are highly desired in biological applications. Here, we report two novel types of visible-light photoswitchable peptide nucleic acids (PNAs) based on the molecular transducers: hemithioindigo and tetra-ortho-fluoroazobenzene. Our study reveals that the tetra-ortho-fluoroazobenzene–PNA conjugates have promising properties (fast reversible isomerization, exceptional thermal stability, high isomer conversions and sensitivity to visible-light irradiation) as reversible modulators to control oligonucleotide hybridization in biological contexts. Furthermore, we verified that this switchable modification delivers a slightly different hybridization behavior in the PNA. Thus, both melting experiments and strand-displacement assays showed that in all the cases the trans-isomer is the one with superior binding affinities. Alternative versions, inspired by our first compounds here reported, may find applications in different fields such as chemical biology, nanotechnology and materials science.

Photo-Control of Small GTPase RAS using Photo Responsive Peptide Inhibitor Which Mimic αH Helix of SOS

Small GTPase Ras is known as a central regulator of several cellular signaling which are related to cell viability, growth, differentiation and apoptosis. Ras is already studied well at molecular level and they have two state, GTP binding (active) state and GDP-binding (inactive) state. These switching of Ras activity was regulated by two factors, GEFs and GAPs. GEFs serves as Ras activator and they replace GDP from Ras and attach GTP to Ras alternatively. While, GAPs serves as Ras inactivator and they accelerate Ras GTPase. Previously it has been reported that SOSαH mimicking peptide which has cyclic structure at N-terminal region exhibited stablea-helix structure, and inhibit Ras GDP-GTP exchange activity competitively with SOS. In this study, we focused on the interaction between Ras and aH-helix which locates Ras interface of SOS (Ras-GEF) in order to photo-regulate Ras function artificially. Several kinds of αH mimicking peptides which have two cysteine to be crosslinked were synthesized. The cysteine residues of the peptides were crosslinked by bifunctional azobenzene derivatives, azobenzene di-maleimide (ABDM) intramolecularly. The ABDM incorporated into the peptides showed cis-trans isomerization upon ultraviolet (UV) light and visible light irradiations. CD spectral analysis revealed that trans-ABDM-αH peptide forms partially ahelix and cis-ABDM-aH peptide exhibit random secondary structure. SOS dependent GDP-GTP exchange reaction of Ras was inhibited by trans- ABDM-aH peptide. Furthermore, Ex vivo experiment revealed the SOS mimicking peptides exhibit cell membrane permeation indicating the photochromic peptides are applicable to regulation of cell function.

A photo-responsive peptide- and asparagine–glycine–arginine (NGR) peptide-mediated liposomal delivery system

The conjugation of tunable peptides or materials with nanocarriers represents a promising approach for drug delivery to tumor cells. In this study, we report the development of a novel liposomal carrier system that exploits the cell surface binding synergism between photo-sensitive peptides (PSPs) and targeting ligands. The positive charges of the lysine residues on the cell-penetrating peptides (CPPs) were temporarily caged by the photolabile-protective groups (PG), thereby forming a PSP. Furthermore, this PSP enhances specific uptake into cancer cells after rapidly uncaging the PG via near-infrared (NIR) light illumination. In the circulatory system, the cell penetrability of PSP was hindered. In contrast, the asparagine–glycine–arginine (NGR) peptide moieties, selectively bind to CD13-positive tumors, were attached to the nanocarrier to facilitate the active accumulation of this liposomal carrier in tumor tissue. The dual-modified liposomes (PSP/NGR-L) were prepared by emulsification method, and the concentrations of DSPE-PEG2000-psCPP and DSPE-PEG5000-NGR in the liposomes were chosen to be 4% and 1% (molar ratio), respectively. The mean particle size of the PSP/NGR-L was about 95 nm, and the drug entrapment efficiency was more than 90%. Cellular uptake results demonstrated that the proposed PSP/NGR-L had an enhancement of cancer cell recognition and specific uptake. Furthermore, the PSP/NGR-L demonstrated a stronger antitumor efficacy in the HT-1080 tumor model in nude mice with the aid of NIR illumination.

In vitro selection of a photoresponsive peptide aptamer to glutathione-immobilized microbeads

Photoresponsive peptide aptamer to glutathione-immobilized microbeads was in vitro selected using ribosome display incorporated with tRNA carrying an amino acid coupled with an azobenzene.

1H, 13C and 15N chemical shift assignments of unliganded Bcl-xL and its complex with a photoresponsive Bak-derived peptide

Here we report the (1)H, (13)C and (15)N resonance assignments of free Bcl-x(L) and of Bcl-x(L) in complex with an azobenzene-modified peptide derived from the BH3 domain of the pro-apoptotic Bak. The spectra suggest predominantly folded proteins; chemical shift difference analysis provides a detailed view of the reorganization occurring on peptide binding.

In vitro selection of a photo-responsive peptide aptamer using ribosome display

A photo-responsive peptide aptamer against microbeads immobilized streptavidin was isolated using in vitro selection combined with photo-manipulation. This is the first example of the introduction of a peptide aptamer in the photo-control of dynamic molecular recognition.

Development and photo-responsive peptide bond cleavage reaction of two-photon near-infrared excitation-responsive peptide

Two-photon near-infrared excitation-responsive amino acid was developed. It was incorporated into a peptide, and focused near-infrared pulsed laser irradiation-induced peptide bond cleavage reaction at the C-terminal position of the photo-responsive amino acid was observed.

RNA Aptamers That Reversibly Bind Photoresponsive Azobenzene‐Containing Peptides

Modulation of biological networks assembled by diverse interactions among biologically active molecules has provided a platform for innovative biotechnologies. Here, we report RNA aptamers that bind to a photoresponsive peptide (KRAzR; Lys-Arg-azobenzene-Arg) containing azobenzene chromophore, which can change its structure by photoirradiation. Aptamers were identified after 10 cycles of an in vitro selection procedure starting with a DNA library containing a 70 nt random region. Surface plasmon resonance (SPR) analysis demonstrated that interactions between aptamers and KRAzR were fully controlled by appropriate photoirradiation to the SPR sensor chip. Upon irradiation of 360 nm on the KRAzR-immobilized surface, the binding of each aptamer to the surface was significantly decreased. Subsequent photoirradiation of the same surface with 430 nm restored the aptamer binding to the surface. We also observed that direct photoirradiation of the aptamer-peptide complex on a gold surface actively promoted dissociation of the complex. Furthermore, a doped reselection method was applied to acquire structural and sequence information of aptamer 66. From a data analysis of the conserved region and the mutation frequency, we were able to select a plausible secondary structure among three candidates predicted by computational folding simulation.

RNA aptamers that reversibly bind to photoresponsive peptide

Modulation of biological network constituted by diverse interactions among biologically active molecules has provided innovative biotechnologies. Here, we report RNA aptamers that bind to photoresponsive peptide (KRAzR; Lys-Arg-Azobenzene-Arg) containing azobenzene chromophore, which can change its geometrical structure by phohtoirradiation. Aptamers were identified by 10 cycles of in vitro selection procedure from DNA library containing 70 nt random region. Surface plasmon resonance (SPR) analysis demonstrated that interactions between aptamers and KRAzR were fully controlled by appropriate photoirradiation. Upon irradiation of 360 nm light over the KRAzR-immobilized surface, the binding of each aptamer to the surface was significantly decreased. Subsequent photoirradiation of the same surface with 430 nm light restored the aptamer binding ability of the surface. We also observed that direct photoirradiation of aptamer-peptide complex on a gold surface actively promoted dissociation of the complex.

Photoresponsive peptide–azobenzene conjugates that specifically interact with platinum surfaces

The aim of this study is to prepare photoresponsive peptide–azobenzene compounds which interacts with platinum surfaces specifically, in order to create smart surfaces for further novel applications in design of smart biosensors and array platforms. Here, a water-soluble azobenzene molecule, 4-hydroxyazo benzene,4-sulfonic acid was synthesized by diazo coupling reaction. A platinum-specific peptide, originally selected by a phage display technique was chemically synthesized/purchased, and conjugated with the azobenzene compound activated with carbonyldiimidazole. Both azobenzene and its conjugate were characterized (including photoresponsive properties) by FTIR, NMR, and UV-spectrophotometer. The yield of conjugation reaction estimated by ninhydrin assay was about 65%. Peptide incorporation did not restrict the light-sensitivity of azobenzene. Adsorption of both the peptide and its azobenzene conjugate was followed by Quartz Crystal Microbalance (QCM) system. The kinetic evaluations exhibited that both molecules interact platinum surfaces, quite rapidly and strongly.

Quadruple Helix Formation of a Photoresponsive Peptide Amphiphile and Its Light-Triggered Dissociation into Single Fibers

Using a peptide amphiphile having a bulky photolabile 2-nitrobenzyl group between the alkyl chain and the peptide segment, we demonstrated quadruple helical fiber formation and its dissociation into single fibrils in response to light. Putting the bulky group close to the core of a fibril is thought to induce a distortion of the alignment of molecules, which can in turn lead to quadruple helices. Photoirradiation to cleave the bulky group transforms the helices into single fibrils.

Reversible regulation of binding between a photoresponsive peptide and its RNA aptamer

It will be important for biologists to artificially and reversibly control gene expression in vivo through the interaction of RNA and small molecules. In this symposium, we report that RNA aptamers obtained from in vitro selection in which a photoresponsive short peptide containing the azobenzene moiety with flanking arginine residues on both sides as a ligand provided reversible binding to the ligand peptide immobilized onto the gold surface. We designed and synthesized a photoresponsive short peptide that can interact with RNA, can convert its conformation reversibly by photoirradiation, and can be produced on a large scale for in vitro selection. The RNA pool contained N70 random sequences, and after the eighth cycle we identified RNA aptamers showing the Kd of about a few microM. A surface plasmon resonance (SPR) experiment revealed that RNA aptamers could bind to the transisomer of the peptide immobilized on the gold surface, but not to the cis-peptide isomerized by photoirradiation with 360 nm light to the gold surface. The SPR signals were recovered after photoirradiation with 430 nm light leading to isomerization of the peptide from cis to trans.

Photoresponsive peptide and polypeptide systems 15*: Synthesis of photo-crosslinkable poly(amino acid)s by watery process and its application as a reinforcement for polyion complex fibers

The present article described the synthesis of the coumarin-containing polypeptides using the water-soluble active ester method and the reinforcement of the polyion complex (PIC) fibers by the photochemical cross-linking between the polypeptide-pendant coumaryloxyacetyl groups. Two kinds of cationic polypeptides, poly(α,L-lysine) (PLL) and poly(α,L-ornithine) (PLO) were treated with (4-hydroxyphenyl)dimethyl sulfonium methyl sulfate ester in water to incorporate the photoreactive coumaryloxyacetyl groups into the side chain amino groups of the PLL and PLO. The contents of the photoreactive groups in the produced coumarin-containing polypepides were controlled by the molar ratios of the amino groups to the active ester. The resulting coumarin-containing cationic polypeptides were spun into the PIC fibers with the anionic polysaccharide gellan in water. The tensile strengths of the coumarin-containing PIC fibers were superior to those of the intact PLL- and PLO-gellan PIC fibers. The mechanical strength of the coumarin-containing PIC fibers further increased upon light-irradiation. From these results, we concluded that the hydrophobic interactions and the covalent cross-linking due to the photochemical dimerization between the coumaryloxyacetyl groups significantly contributed to reinforcement of the PIC fibers. The present results afford a new methodology to the PIC reinforcement that was achieved by the polymer modification reaction in watery process.

Photomodulation of conformational states. II. Mono- and bicyclic peptides with (4-aminomethyl)phenylazobenzoic acid as backbone constituent.

It has been reported that backbone cyclization of octapeptides with the photoresponsive (4-aminomethyl)phenylazobenzoic acid imparts sufficient restraints to induce and stabilize ordered conformations of the peptide backbone in both the cis- and trans-azo-isomers (L. Ulysse, J. Cubillos, and J. Chmielewski, Journal of the American Chemical Society, 1995, Vol. 117, pp. 8466-8467). Correspondingly, the active-site octapeptide fragment H-Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-OH [134-141] of thioredoxin reductase, with its high preference for a 3(10)-helix turn conformation centered on the Thr-Cys sequence, was backbone cyclized with this azobenzene moiety in the attempt to design a photoresponsive system where the conformational states of the peptide backbone are dictated by the configuration of the azobenzene and can be further modulated by the disulfide bridge. Nuclear magnetic resonance conformational analysis of the monocyclic compound clearly revealed the presence of two conformational families in both the cis- and trans-azo configuration. Of the higher populated conformational families, the structure of the trans-isomer seems like a pretzel-like folding, while the cis-isomer relaxes into a significantly less defined conformational state that does not exhibit any regular structural elements. Further restrictions imparted by disulfide bridging of the peptide moiety leads to an even better defined conformation for the trans-azo-isomer, whereas the cis-isomer can be described as a frustrated system without pronounced energy minima and thus with little conformational preferences. Our findings would suggest that this photoresponsive peptide template may not be of general usefulness for light-induced conformational transitions between two well-defined conformational states at least under the experimental conditions employed, even in the bicyclic form. However, trans --> cis isomerization of the bicyclic peptide is accompanied by a switch from a well-defined conformation to an ensemble of possible conformations.

Photoresponsive Peptide and Polypeptide Systems. 13. Photoinduced Cross-Linked Gel and Biodegradation Properties of Copoly(l-lysine) Containing ε-7-Coumaryloxyacetyl-l-lysine Residues

Copoly[LysLys(Cou)] containing e-7-coumaryloxyacetyl-l-lysine [Lys(Cou)] residues were synthesized by the N-carboxyanhydride method. When the aqueous solutions of copoly[LysLys(Cou)] containing 5−10 mol % of Lys(Cou) are irradiated by light, the photo-cross-linking reaction proceeds slowly between coumarin moieties in the side chains to give a cis head-to-head cyclocoumarin, and after 6−24 h the solutions become transparent hydrogels. The gels exhibit solvent-induced reversible expansion and contraction behavior in water and in ethanol. The biodegradation of the hydrogels by some proteolytic enzymes and soil filamentous fungi has been investigated using photo-cross-linked copoly[LysLys(Cou)] gels. The photo-cross-linked copoly[Lys90-95Lys(Cou)10-5] gels are degradable by trypsin and protease type XXIII or two microorganisms, Aspergillus oryzae and Penicillium citrinum, suggesting potential for a controlled biodegradation by the different cross-linking densities in the gel matrixes upon the controlled irra...

Photoresponsive peptide and polypeptide systems. Part 7. Reversible chiral photochromism and solubility change of azo aromatic L-lysine related compounds

Disubstituted azo aromatic L- and D-lysine homologues with one to four methylene groups in the side chain, and related compounds, have been synthesized. The photochemical properties of the azo aromatic lysine series have been studied from both achiral and chiral standpoints. On irradiation at different wavelengths, the azo aromatic lysine series change their solubilities achirally in certain solvents, being soluble under u.v. light (cis) and precipitating out under visible light (trans). The dipole moments of the solvents used are considered to be important in this solubility change. The chiral photochemical properties of the above lysine related compounds have been examined by absorption and circular dichroism (c.d.) spectroscopy. The whole c.d. spectrum exhibits reversible photochromism on irradiation with light, owing to the trans-cis photoisomerization of the azo aromatic moieties. The exciton chirality method has been successfully applied in order to demonstrate the origin of the dichroic bands in the 260–390 nm wavelength region which exhibit coupling of the transition dipole moment due to the transπ-π* transition of the azo aromatic chromophore under an asymmetric Cα carbon environment.

Photoresponsive Peptide and Polypeptide Systems. VI. Reversible Solubility Change of Azo Aromatic Lysine

Abstract The reversible solubility change of azo aromatic lysines due to the trans–cis photoconversion of azo moieties was studied. On irradiation at different wavelengths, azo aromatic lysines are soluble under ultraviolet light (cis) and precipitate under visible light (trans). The dipole moments of the solvents used are considered to play a role in the solubility change.