Protein Affinity Reagents Research Articles

Multimodal Affinity-Modulated Efficient Separation of Lysozyme with a Hierarchical MXene@MOF Hybrid Framework.

The development of abiotic protein affinity adsorbents remains challenging for the accurate acquisition and analysis of specific protein species. Inspired by bacterial cell walls, a hierarchical hybrid framework is fabricated through the oriented growth of an Fe-based metal organic framework (MOF) on V2C MXene for the efficient separation of lysozyme (Lys). After directed evolution of adsorptive materials, the MXene@MOF composite rich in hydroxyl groups (termed as MX@MOF-DH) is found exerting exceptional affinity for Lys. Benefiting from hydrogen-bonding, coordination, and electrostatic interaction-mediated multimodal and multivalent affinity, MX@MOF-DH reveals rapid adsorption rate (5 min), superb enrichment factor (83.1), and favorable binding capacity (609.7 mg g-1), which outperforms other latest adsorbents. Moreover, femtomolar sensitivity is achieved even in the presence of high-abundant interfering proteins, as confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis. This work not only provides an efficient approach for selective enrichment of lysozyme but also paves an avenue to construct the protein affinity reagents for specific biological medicine and analysis applications.

Revisiting Protein-Copolymer Binding Mechanisms: Insights beyond the "Lock-and-Key" Model.

The "lock-and-key" model that emphasizes the concept of chemical-structural complementary is the key mechanism for explaining the selectivity between small ligands and a larger adsorbent molecule. In this work, concerning the copolymer chain using only the combination of N-isopropylacrylamide (NIPAm) and hydrophobic N-tert-butylacrylamide (TBAm) monomers and by large-scale atomistic molecular dynamics simulations, our results show that the flexible copolymer chain may exhibit strong binding affinity for the biomarker protein epithelial cell adhesion molecule, in the absence of hydrophobic matching and strong structural complementarity. This surprising binding behavior, which cannot be anticipated by the "lock-and-key" model, can be attributed to the preferential interactions established by the copolymer with the protein's hydrophilic exterior. We observe that increasing the fraction of incorporated TBAm monomers leads to a prevalence of interactions with asparagine and glutamine amino acids due to the emerging hydrogen bonding with both NIPAm and TBAm monomers. Our findings suggest the appearance of highly specific and high-affinity binding sites on the protein created by engineering the copolymer composition, which motivates the applications of copolymers as protein affinity reagents.

Abiotic Synthetic Antibodies to Target a Specific Protein Domain and Inhibit Its Function.

The Bacillus thuringiensis (Bt) Cry proteins are widely used in insect pest control. Despite their economic benefits, remaining concerns over potential ecological and health risks warrant their ongoing surveillance. Affinity reagents, most often antibodies, protein scaffolds, and aptamers, are the traditional tools used for protein binding and detection. We report a synthetic antibody (SA) alternative to traditional biological affinity reagents for binding Bt Cry proteins. Analysis of hotspots of the Bt Cry protein-insect midgut cadherin-like receptor complexes was used for the design of the SA. The SA was selected from a small focused library of hydrogel copolymers containing functional monomers complementary to key exposed hotspots of Bt Cry proteins. A directed chemical evolution identified a SA, APhe-NP23, with affinity and selectivity for Bt Cry1Ab/Ac proteins. The putative intermolecular polymer-protein interfaces were identified by the SA's uptake of Bt Cry1Ac pepsin hydrolysates, binding epitope mutation studies, and protein-protein inhibition studies of the toxin binding to its native insect receptor binding domains. The SA inhibitor binds to the same protein domains as the insect's cadherin-like receptors, Bt-R1 and SeCad1b. The SA binds rapidly to Bt Cry1Ab/Ac with high capacity, is pH-responsive, and is synthesized reproducibly. We believe that a hotspot-directed approach is general for creation of abiotic protein affinity reagents that target functional protein domains. Affinity ligands are typically high-information content biologicals. Their structure and function are determined from their amino acid or oligo sequence. In contract, the SA described in this work is a statistical copolymer that lacks sequence specificity. These results are an important contribution to the concept that randomness and biospecificity are not mutually exclusive.

A stepwise mutagenesis approach using histidine and acidic amino acid to engineer highly pH-dependent protein switches.

Antibody-based drugs can be highly toxic, because they target normal tissue as well as tumor tissue. The pH value of the extracellular microenvironments around tumor tissues is lower than that of normal tissues. Therefore, antibodies that engage in pH-dependent binding at slightly acidic pH are crucial for improving the safety of antibody-based drugs. Thus, we implemented a stepwise mutagenesis approach to engineering pH-dependent antibodies capable of selective binding in the acidic microenvironment in this study. The first step involved single-residue histidine scanning mutagenesis of the antibody's complementarity-determining regions to prescreen for pH-dependent mutants and identify ionizable sensitive hot-spot residues that could be substituted by acidic amino acids to obtain pH-dependent antibodies. The second step involved single-acidic amino acid residue substitutions of the identified residues and the assessment of pH-dependent binding. We identified six ionizable sensitive hot-spot residues using single-histidine scanning mutagenesis. Nine pH-dependent antibodies were isolated using single-acidic amino acid residue mutagenesis at the six hot-spot residue positions. Relative to wild-type anti-CEA chimera antibody, the binding selectivity of the best performing mutant was improved by approximately 32-fold according to ELISA and by tenfold according to FACS assay. The mutant had a high affinity in the pH range of 5.5–6.0. This study supports the development of pH-dependent protein switches and increases our understanding of the role of ionizable residues in protein interfaces. The stepwise mutagenesis approach is rapid, general, and robust and is expected to produce pH-sensitive protein affinity reagents for various applications.

Design of Synthetic Polymer Nanoparticles That Capture and Neutralize Target Molecules

Protein affinity reagents that specifically and strongly bind to target molecules are widely used in disease detection, diagnosis, and therapy. Although antibodies and their fragments are the gold standard in protein-protein inhibitors (PPIs), synthetic polymers such as linear polymers, dendrimers, and nanoparticles as cost-effective PPIs have attracted great attention as alternatives to antibodies. These polymers exhibit high affinity to the target by imitating natural protein-protein interactions. However, only a few in vivo applications have been reported. Here, our recent advances in the development of synthetic polymers for in vivo application are reviewed. Poly(N-isopropylacrylamide) (pNIPAm) was used as a model of synthetic affinity reagents. Incorporation of both sulfated carbohydrate and hydrophobic monomers into lightly crosslinked pNIPAm nanoparticles (NPs) captured and neutralized vascular endothelial growth factor (VEGF) and inhibited tumor growth upon intravenous injection into tumor-bearing mice. Modification of a liposome with the pNIPAm-based linear polymer increased the polymer circulation time after intravenous injection and improved the affinity for the target. The pNIPAm-based NPs delivered by oral administration captured the target small molecules and inhibited their absorption from the intestine. Our recent findings provide useful information for the design of synthetic polymers that capture target molecules in vivo.

Abiotic Stimuli-Responsive Protein Affinity Reagent for IgG.

We describe an approach for the discovery of protein affinity reagents (PARs). Abiotic synthetic hydrogel copolymers can be "tuned" for selective protein capture by the type and ratios of functional monomers included in their polymerization and by the polymerization conditions (i.e., pH). By screening libraries of hydrogel nanoparticles (NPs) containing charged and hydrophobic groups against a protein target (IgG), a stimuli-responsive PAR is selected. The robust carbon backbone synthetic copolymer is rapidly synthesized in the chemistry laboratory from readily available monomers. The production of the PAR does not require living cells and is free from biological contamination. The capture and release of the protein by the copolymer NP is reversible. IgG is sequestered from human serum at pH 6.5 and following a wash step, the purified protein is released by elevating the pH to 7.3. The binding and release of the protein occur without denaturation. The abiotic material functions as a selective PAR for the F(ab')2 domain of IgG for pull-down and immunoprecipitation experiments and for isolation and purification of proteins from complex biological mixtures.

Design of synthetic polymer nanoparticles that inhibit glucose absorption from the intestine

Synthetic polymers prepared using several functional monomers have attracted attention as cost-effective protein affinity reagents and alternative to antibodies. We previously reported the synthesis of poly NIPAm-based nanoparticles (NPs) using several functional monomers that can capture target molecules. In this study, we designed NPs for capturing glucose and inhibiting intestinal absorption in living mice. For capturing glucose, we focused on the Maillard reaction between primary amines and aldehyde residues. We hypothesized that the primary amine-containing NPs can capture the open-chain structure of glucose via the Maillard reaction and inhibit intestinal absorption. NPs were prepared by the precipitation polymerization of NIPAm, N-tert-butylacrylamide (TBAm), trifluoroacetate-protected N-(3-aminopropyl)methacrylamide (T-APM), and N,N′-methylenebisacrylamide. Then, T-APM in NPs was deprotected by NH3 (aq). The amount of glucose captured by NPs depended on the percentage of TBAm and APM in vitro. After 24 h, only 2% of orally administered NPs remained in the body after administration, suggesting that many NPs were excreted without being absorbed. The prepared NPs significantly inhibited an increase in blood glucose concentration after the oral administration of glucose and NPs, indicating that NPs capture glucose and inhibit intestinal absorption. These results show the potential of using synthetic polymer nanoparticles for inhibiting postprandial hyperglycemia.

Design of Functional Nanoparticles for Intractable Disease Therapy.

Protein affinity reagents are widely used for basic research, diagnostics, and disease therapy. Antibodies and their fragments are known as the most common protein affinity reagents. They specifically and strongly bind to target molecules and inhibit their functions. Thus, antibody drugs have increased in the recent two decades for disease therapy, such as cancer. These strong protein-protein interactions are composed of a nexus of multiple weak interactions. Synthetic polymers that bind to target molecules have been developed by the imitation of protein-protein interactions. These polymers show nanomolar affinity for the target and neutralize their functions; thus, they are of significant interest as a cost-effective protein affinity reagent. We have been developing synthetic polymer nanoparticles (NPs) that bind to target peptides and proteins by the inclusion of several functional monomers, such as charged and hydrophobic monomers. In this review, the focus is on the design of synthetic polymer NPs that bind to target molecules for disease therapy. We succeeded in neutralization of toxic peptides and signaling proteins both in vitro and in vivo. Additionally, linear polymers were modified on a lipid nanoparticle surface to improve polymer biodistribution. Our recent findings should provide useful information for the development of abiotic protein affinity reagents.

Engineering of Lipid Nanoparticles by the Multifunctionalization of the Surface with Amino Acid Derivatives for the Neutralization of a Target Toxic Peptide

AbstractProtein affinity reagents (e.g., antibodies) are often used for basic research, diagnostics, separations, and disease therapy. Although a lot of “synthetic” protein affinity reagents have been developed as a cost‐effective alternative to antibodies, their low biocompatibility is a considerable problem for clinical application. Lipid nanoparticles (LNP) represent a highly biocompatible drug delivery agent. However, little has been reported that LNP itself works as a protein affinity reagent in living animals. Here, LNP is engineered for binding to and neutralizing a target toxic peptide in living animals by multifunctionalization with amino acid derivatives. Multifunctionalized LNP (MF‐LNP) is prepared using amino acid derivative‐conjugated lipids. Optimized MF‐LNP exhibits nanomolar affinity to the target toxic peptide and inhibits toxic peptide‐dependent hemolysis and cytotoxicity. In addition, MF‐LNP captures and neutralizes the toxic peptide after intravenous injection in the bloodstream; in addition, MF‐LNP does not release the toxic peptide in the accumulated organ. These results reveal the potential of using LNP as a highly biocompatible protein affinity reagent such as an antidote.

Antibody validation for Western blot: By the user, for the user

Antibody validation for Western blot: By the user, for the user

High-Throughput Quantification of Surface Protein Internalization and Degradation.

Cell surface proteins are key regulators of fundamental cellular processes and, therefore, often at the root of human diseases. Thus, a large number of targeted drugs which are approved or under development act upon cell surface proteins. Although down-regulation of surface proteins by many natural ligands is well-established, the ability of drug candidates to cause internalization or degradation of the target is only recently moving into focus. This property is important both for the pharmacokinetics and pharmacodynamics of the drug but may also constitute a potential resistance mechanism. The enormous numbers of drug candidates targeting cell surface molecules, comprising small molecules, antibodies, or alternative protein scaffolds, necessitate methods for the investigation of internalization and degradation in high throughput. Here, we present a generic high-throughput assay protocol, which allows the simultaneous and independent quantification of internalization and degradation of surface proteins on a single-cell level. Because we fuse a HaloTag to the cell surface protein of interest and exploit the differential cell permeability of two fluorescent HaloTag ligands, no labeling of the molecules to be screened is required. In contrast to previously described approaches, our homogeneous assay is performed with adherent live cells in a 96-well format. Through channel rescaling, we are furthermore able to obtain true relative abundances of surface and internal protein. We demonstrate the applicability of our procedure to three major drug targets, EGFR, HER2, and EpCAM, examining a selection of well-investigated but also novel small molecule ligands and protein affinity reagents.

Sequestering and inhibiting a vascular endothelial growth factor in vivo by systemic administration of a synthetic polymer nanoparticle

Protein affinity reagents (PARs), frequently antibodies, are essential tools for basic research, diagnostics, separations and for clinical applications. However, there is growing concern about the reproducibility, quality and cost of recombinant and animal-derived antibodies. This has prompted the development of alternatives that could offer economic, and time-saving advantages without the use of living organisms. Synthetic copolymer nanoparticles (NPs), engineered with affinity for specific protein targets, are potential alternatives to PARs. Although there are now a number of examples of abiotic protein affinity reagents (APARs), most have been evaluated in vitro limiting a realistic assessment of their potential for more demanding, practical in vivo applications. We demonstrate for the first time that an abiotic copolymer hydrogel nanoparticle (NP1) engineered to bind a key signaling protein, vascular endothelial growth factor (VEGF165), functions in vivo to suppress tumor growth by regulating angiogenesis. Lightly cross-linked N-isopropylacrylamide based NPs that incorporate both sulfated N-acetylglucosamine and hydrophobic monomers were optimized by dynamic chemical evolution for VEGF165 affinity. NP1 efficacy in vivo was evaluated by systemic administration to tumor-bearing mice. The study found that NP1 suppresses tumor growth and reduces tumor vasculature density. Combination therapy with doxorubicin resulted in increased doxorubicin concentration in the tumor and dramatic inhibition of tumor growth. NP1 treatment did not show off target anti-coagulant activity. In addition, >97% of injected NPs are rapidly excreted from the body following IV injection. These results establish the use of APARs as inhibitors of protein-protein interactions in vivo and may point the way to their broader use as abiotic, cost effective protein affinity reagents for the treatment of certain cancers and more broadly for regulating signal transduction.

A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors.

A key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org.

Modular design of optically controlled protein affinity reagents.

Photopharmaceuticals can, in principle, be created by linking photoswitchable moieties to bioactive molecules. However, a general strategy for converting a therapeutic agent into its photoswitchable version is not currently available. Herein we propose a generalizable, modular approach for obtaining light controllable bioactive agents by modifying the scaffold of a protein affinity reagent using an azobenzene photoswitch.

A polymer nanoparticle with engineered affinity for a vascular endothelial growth factor (VEGF165).

Protein affinity reagents are widely used in basic research, diagnostics and separations and for clinical applications, the most common of which are antibodies. However, they often suffer from high cost, and difficulties in their development, production and storage. Here we show that a synthetic polymer nanoparticle (NP) can be engineered to have many of the functions of a protein affinity reagent. Polymer NPs with nM affinity to a key vascular endothelial growth factor (VEGF165) inhibit binding of the signalling protein to its receptor VEGFR-2, preventing receptor phosphorylation and downstream VEGF165-dependent endothelial cell migration and invasion into the extracellular matrix. In addition, the NPs inhibit VEGF-mediated new blood vessel formation in Matrigel plugs in vivo. Importantly, the non-toxic NPs were not found to exhibit off-target activity. These results support the assertion that synthetic polymers offer a new paradigm in the search for abiotic protein affinity reagents by providing many of the functions of their protein counterparts.

Highlights of the Biology and Disease-driven Human Proteome Project, 2015-2016.

The Biology and Disease-driven Human Proteome Project (B/D-HPP) is aimed at supporting and enhancing the broad use of state-of-the-art proteomic methods to characterize and quantify proteins for in-depth understanding of the molecular mechanisms of biological processes and human disease. Based on a foundation of the pre-existing HUPO initiatives begun in 2002, the B/D-HPP is designed to provide standardized methods and resources for mass spectrometry and specific protein affinity reagents and facilitate accessibility of these resources to the broader life sciences research and clinical communities. Currently there are 22 B/D-HPP initiatives and 3 closely related HPP resource pillars. The B/D-HPP groups are working to define sets of protein targets that are highly relevant to each particular field to deliver relevant assays for the measurement of these selected targets and to disseminate and make publicly accessible the information and tools generated. Major developments are the 2016 publications of the Human SRM Atlas and of "popular protein sets" for six organ systems. Here we present the current activities and plans of the BD-HPP initiatives as highlighted in numerous B/D-HPP workshops at the 14th annual HUPO 2015 World Congress of Proteomics in Vancouver, Canada.

Proteomic study of hepatocellular carcinoma using a novel modified aptamer-based array (SOMAscan™) platform

Vascular invasion is a pathological hallmark of hepatocellular carcinoma (HCC), associated with poor prognosis; it is strongly related to the early recurrence and poor survival after curative resection. In order to determine the proteomic backgrounds of HCC carcinogenesis and vascular invasion, we employed a novel modified aptamer-based array (SOMAscan) platform. SOMAscan is based on the Slow Off-rate Modified Aptamers (SOMAmers), which rely on the natural 3D folding of single-stranded DNA-based protein affinity reagents. Currently, the expression level of 1129 proteins can be assessed quantitatively. Correlation matrix analysis showed that the overall proteomic features captured by SOMAscan differ between tumor and non-tumor tissues. Non-tumor tissues were shown to have more homogeneous proteome backgrounds than tumor tissues. A comparative study identified 68 proteins with differential expression between tumor and non-tumor tissues, together with eight proteins associated with vascular invasion. Gene Ontology analysis showed that the extracellular space and extracellular region proteins were predominantly detected. Network analysis revealed the linkage of seven proteins, AKT1, MDM2, PTEN, FGF1, MAPK8, PRKCB, and FN1, which were categorized as the components of “Pathways in cancer” in pathway analysis. The results of SOMAscan analysis were not concordant with those obtained by western blotting; only the determined FN1 levels were concordant between the two platforms. We demonstrated that the proteome captured by SOMAscan includes the proteins relevant to carcinogenesis and vascular invasion in HCC. The identified proteins may serve as candidates for the future studies of disease mechanisms and clinical applications.

Abstract 3889: An aptamer-based multiplexed proteomic technology reveals differential PKC ligand responses

Abstract Bryostatin 1 (Bryo 1) is in clinical trials for cancer and Alzheimer's disease. Whereas Bryo 1, like the phorbol esters, binds to and activates protein kinase C (PKC), it paradoxically antagonizes many but not all phorbol ester responses. The mechanistic basis for this differential response is still unknown. In the LNCaP human prostate cancer cell line, the typical phorbol ester PMA causes growth inhibition whereas Bryo 1 does not. Previously, we showed that PMA induces dramatic changes in gene transcription in the LNCaP cells as determined by gene expression microarray analysis. The effects of Bryo 1 on gene expression were initially similar at 1 hr but were variably more transient, as seen at 6 hr, depending on the specific gene. We think that this transiency of action is a critical mechanistic feature of Bryo 1. The current study extends to the comparison of the effects of Bryo 1 and PMA on the level of global protein expression. Using a new generation of protein-capture SOMAmer (Slow Off-rate Modified Aptamer) reagents, the SOMAscan (Somalogic, Boulder, CO) proteomic assay simultaneously measures over one thousand analytes within a minute amount of protein sample. SOMAmer reagents are chemically modified single stranded DNA-based protein affinity reagents that recognize specific conformational epitopes of native 3D proteins with good specificity and dynamic range. The technology is emerging as a highly sensitive, multiplexed and quantitative proteomic tool for biomarker discovery and validation. 1,129 targets (covering major gene families including receptors, kinases, growth factors, and a diverse collection of secreted, intracellular and extracellular proteins or domains) were evaluated in both total cell lysate and nuclear extracts of LNCaP cells treated with DMSO, PMA and Bryo 1 for 60 minute and 6 hours. Compared to the DMSO control, the lysates of cells treated for 60 minute with either PMA and Bryo 1 had over three hundred targets that showed significant changes (p<0.01). While a similar scale of signaling response was observed in the 6 hour PMA treated total cell lysate, less than 50 targets differed from the DMSO control in the 6 hour Bryo 1 treated samples (p<0.01). Similar results were obtained for the nuclear extracts. Good assay reproducibility was observed within replicates. While the protein analysis confirmed the transient nature of Bryo 1 action, comparison of the SOMAscan protein expression data with the mRNA transcript analysis revealed substantial differences, indicating the importance of monitoring signaling response at both the transcriptomic and proteomic levels. It further identified a rich set of protein targets involved in the differential PKC signaling by different ligands. On-going studies seek to characterize the basis for the transient signaling response following Bryo 1 treatment. Citation Format: Jinqiu Chen, Noemi Kedei, David J. Goldstein, Mariam Q. Malik, Peter M. Blumberg. An aptamer-based multiplexed proteomic technology reveals differential PKC ligand responses. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3889.

Tuning the Protein Corona of Hydrogel Nanoparticles: The Synthesis of Abiotic Protein and Peptide Affinity Reagents.

Nanomaterials, when introduced into a complex, protein-rich environment, rapidly acquire a protein corona. The type and amount of proteins that constitute the corona depend significantly on the synthetic identity of the nanomaterial. For example, hydrogel nanoparticles (NPs) such as poly(N-isopropylacrylamide) (NIPAm) have little affinity for plasma proteins; in contrast, carboxylated poly(styrene) NPs acquire a dense protein corona. This range of protein adsorption suggests that the protein corona might be "tuned" by controlling the chemical composition of the NP. In this Account, we demonstrate that small libraries of synthetic polymer NPs incorporating a diverse pool of functional monomers can be screened for candidates with high affinity and selectivity to targeted biomacromolecules. Through directed synthetic evolution of NP compositions, one can tailor the protein corona to create synthetic organic hydrogel polymer NPs with high affinity and specificity to peptide toxins, enzymes, and other functional proteins, as well as to specific domains of large proteins. In addition, many NIPAm NPs undergo a change in morphology as a function of temperature. This transformation often correlates with a significant change in NP-biomacromolecule affinity, resulting in a temperature-dependent protein corona. This temperature dependence has been used to develop NP hydrogels with autonomous affinity switching for the protection of proteins from thermal stress and as a method of biomacromolecule purification through a selective thermally induced catch and release. In addition to temperature, changes in pH or buffer can also alter a NP protein corona composition, a property that has been exploited for protein purification. Finally, synthetic polymer nanoparticles with low nanomolar affinity for a peptide toxin were shown to capture and neutralize the toxin in the bloodstream of living mice. While the development of synthetic polymer alternatives to protein affinity reagents is in its early stages, these recent successes using only small libraries of functional monomers are most encouraging. It is likely that by expanding the chemical diversity of functional hydrogels and other polymers, a much broader range of NP-biomacromolecule affinity pairs will result. Since these robust, nontoxic polymers are readily synthesized in the chemistry laboratory, we believe the results presented in this Account offer a promising future for the development of low cost alternatives to more traditional protein affinity reagents such as antibodies.

Photocontrol of Protein Affinity Reagents by Red Light using Azobenzene Switches

Remote control of protein function by light is a powerful technique for manipulating biological processes in living organisms. One general method to obtain photoswitchable proteins is to couple the photoisomerization of azobenzene derivatives to conformational changes in the protein of interest. The applicability of these compounds in vivo, however, depends largely on the excitation wavelength that is required for their switching. Short wavelengths of visible light are highly scattered and do not penetrate well in cells and live tissues. In the past few years, novel azo derivatives which operate with red wavelengths and are stable in water were designed in our group. We have now applied these compounds to fynomers: small proteins based on Fyn SH3 scaffold that are developed via phage display selection techniques to target biologically relevant proteins. We chose a fynomer that was optimized for inhibiting the activity of human chymase by binding near its active site. Chymase is a serine protease that is secreted by mast cells and is shown to be involved in cardiovascular diseases as well as pathological inflammatory conditions. Two cysteine residues were introduced by point mutations in the sequence of the fynomer. Subsequent crosslinking of the inhibitor with azobenzenes at those residues allows only the cis isomer of the azo moiety to be compatible with the well-folded inhibitor. The dark-adapted (trans azo) crosslinked fynomer was partially unfolded and showed reduced inhibitory activity. Upon its irradiation with red light (635 nm), the inhibitor was largely folded and better suppressed the activity of chymase. Since the Fyn SH3 scaffold can be broadly utilized to target virtually any protein, these results demonstrate the promise of red light switchable azobenzenes for in vivo functional studies and photopharmacology.