Stretchable and Bioadhesive Gelatin Methacryloyl-Based Hydrogels Enabled by in Situ Dopamine Polymerization.

Hydrogel patches with high toughness, stretchability, and adhesive properties are critical to healthcare applications including wound dressings and wearable devices. Gelatin methacryloyl (GelMA) provides a highly biocompatible and accessible hydrogel platform. However, low tissue adhesion and poor mechanical properties of cross-linked GelMA patches (i.e., brittleness and low stretchability) have been major obstacles to their application for sealing and repair of wounds. Here, we show that adding dopamine (DA) moieties in larger quantities than those of conjugated counterparts to the GelMA prepolymer solution followed by alkaline DA oxidation could result in robust mechanical and adhesive properties in GelMA-based hydrogels. In this way, cross-linked patches with ∼140% stretchability and ∼19 000 J/m3 toughness, which correspond to ∼5.7 and ∼3.3× improvement, respectively, compared to that of GelMA controls, were obtained. The DA oxidization in the prepolymer solution was found to play an important role in activating adhesive properties of cross-linked GelMA patches (∼4.0 and ∼6.9× increase in adhesion force under tensile and shear modes, respectively) due to the presence of reactive oxidized quinone species. We further conducted a parametric study on the factors such as UV light parameters, the photoinitiator type (i.e., lithium phenyl-2,4,6-trimethylbenzoylphosphinate, LAP, versus 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, Irgacure 2959), and alkaline DA oxidation to tune the cross-linking density and thereby hydrogel compliance for better adhesive properties. The superior adhesion performance of the resulting hydrogel along with in vitro cytocompatibility demonstrated its potential for use in skin-attachable substrates.

Chemical profiling of DNA G-quadruplex-interacting proteins in live cells.

DNA-protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential to understand the underlying regulatory mechanisms on a molecular level. Here we describe a co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in native chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We first showed the robustness of CMPP by proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we then identified hundreds of putative G4-interacting proteins from various functional classes. Next, we confirmed a high G4-binding affinity and selectivity for several newly discovered G4 interactors in vitro, and we validated direct G4 interactions for a functionally important candidate in cellular chromatin using an independent approach. Our studies provide a chemical strategy to map protein interactions of specific nucleic acid features in living cells.

Transepithelial versus epithelium-off corneal crosslinking for progressive keratoconus.

BACKGROUND: Keratoconus is the most common corneal dystrophy. It can cause loss of uncorrected and best-corrected visual acuity through ectasia (thinning) of the central or paracentral cornea, irregular corneal scarring, or corneal perforation. Disease onset usually occurs in the second to fourth decade of life, periods of peak educational attainment or career development. The condition is lifelong and sight-threatening. Corneal collagen crosslinking (CXL) using ultraviolet A (UVA) light applied to the cornea is the only treatment that has been shown to slow progression of disease. The original, more widely known technique involves application of UVA light to de-epithelialized cornea, to which a photosensitizer (riboflavin) is added topically throughout the irradiation process. Transepithelial CXL is a recently advocated alternative to the standard CXL procedure, in that the epithelium is kept intact during CXL. Retention of the epithelium offers the putative advantages of faster healing, less patient discomfort, faster visual rehabilitation, and less risk of corneal haze. OBJECTIVES: To assess the short- and long-term effectiveness and safety of transepithelial CXL compared with epithelium-off CXL for progressive keratoconus. SEARCH METHODS: To identify potentially eligible studies, we searched the Cochrane Central Register of Controlled Trials (CENTRAL) (which contains the Cochrane Eyes and Vision Trials Register) (2020, Issue 1); Ovid MEDLINE; Embase.com; PubMed; Latin American and Caribbean Health Sciences Literature database (LILACS); ClinicalTrials.gov; and World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP). We did not impose any date or language restrictions. We last searched the electronic databases on 15 January 2020. SELECTION CRITERIA: We included randomized controlled trials (RCTs) in which transepithelial CXL had been compared with epithelium-off CXL in participants with progressive keratoconus. DATA COLLECTION AND ANALYSIS: We used standard Cochrane methodology. MAIN RESULTS: We included 13 studies with 723 eyes of 578 participants enrolled; 13 to 119 participants were enrolled per study. Seven studies were conducted in Europe, three in the Middle East, and one each in India, Russia, and Turkey. Seven studies were parallel-group RCTs, one study was an RCT with a paired-eyes design, and five studies were RCTs in which both eyes of some or all participants were assigned to the same intervention. Eleven studies compared transepithelial CXL with epithelium-off CXL in participants with progressive keratoconus. There was no evidence of an important difference between intervention groups in maximum keratometry (denoted 'maximum K' or 'Kmax'; also known as steepest keratometry measurement) at 12 months or later (mean difference (MD) 0.99 diopters (D), 95% CI -0.11 to 2.09; 5 studies; 177 eyes; I2 = 41%; very low certainty evidence). Few studies described other outcomes of interest. The evidence is very uncertain that epithelium-off CXL may have a small (data from two studies were not pooled due to considerable heterogeneity (I2 = 92%)) or no effect on stabilization of progressive keratoconus compared with transepithelial CXL; comparison of the estimated proportions of eyes with decreases or increases of 2 or more diopters in maximum K at 12 months from one study with 61 eyes was RR 0.32 (95% CI 0.09 to 1.12) and RR (non-event) 0.86 (95% CI 0.74 to 1.00), respectively (very low certainty). We did not estimate an overall effect on corrected-distance visual acuity (CDVA) because substantial heterogeneity was detected (I2 = 70%). No study evaluated CDVA gain or loss of 10 or more letters on a logarithm of the minimum angle of resolution (logMAR) chart. Transepithelial CXL may result in little to no difference in CDVA at 12 months or beyond. Four studies reported that either no adverse events or no serious adverse events had been observed. Another study noted no change in endothelial cell count after either procedure. Moderate certainty evidence from 4 studies (221 eyes) found that epithelium-off CXL resulted in a slight increase in corneal haze or scarring when compared to transepithelial CXL (RR (non-event) 1.07, 95% CI 1.01 to 1.14). Three studies, one of which had three arms, compared outcomes among participants assigned to transepithelial CXL using iontophoresis versus those assigned to epithelium-off CXL. No conclusive evidence was found for either keratometry or visual acuity outcomes at 12 months or later after surgery. Low certainty evidence suggests that transepithelial CXL using iontophoresis results in no difference in logMAR CDVA (MD 0.00 letter, 95% CI -0.04 to 0.04; 2 studies; 51 eyes). Only one study examined gain or loss of 10 or more logMAR letters. In terms of adverse events, one case of subepithelial infiltrate was reported after transepithelial CXL with iontophoresis, whereas two cases of faint corneal scars and four cases of permanent haze were observed after epithelium-off CXL. Vogt's striae were found in one eye after each intervention. The certainty of the evidence was low or very low for the outcomes in this comparison due to imprecision of estimates for all outcomes and risk of bias in the studies from which data have been reported. AUTHORS' CONCLUSIONS: Because of lack of precision, frequent indeterminate risk of bias due to inadequate reporting, and inconsistency in outcomes measured and reported among studies in this systematic review, it remains unknown whether transepithelial CXL, or any other approach, may confer an advantage over epithelium-off CXL for patients with progressive keratoconus with respect to further progression of keratoconus, visual acuity outcomes, and patient-reported outcomes (PROs). Arrest of the progression of keratoconus should be the primary outcome of interest in future trials of CXL, particularly when comparing the effectiveness of different approaches to CXL. Furthermore, methods of assessing and defining progressive keratoconus should be standardized. Trials with longer follow-up are required in order to assure that outcomes are measured after corneal wound-healing and stabilization of keratoconus. In addition, perioperative, intraoperative, and postoperative care should be standardized to permit meaningful comparisons of CXL methods. Methods to increase penetration of riboflavin through intact epithelium as well as delivery of increased dose of UVA may be needed to improve outcomes. PROs should be measured and reported. The visual significance of adverse outcomes, such as corneal haze, should be assessed and correlated with other outcomes, including PROs.

Streamlined Target Deconvolution Approach Utilizing a Single Photoreactive Chloroalkane Capture Tag.

Identification of physiologically relevant targets for lead compounds emerging from drug discovery screens is often the rate-limiting step toward understanding their mechanism of action and potential for undesired off-target effects. To this end, we developed a streamlined chemical proteomic approach utilizing a single, photoreactive cleavable chloroalkane capture tag, which upon attachment to bioactive compounds facilitates selective isolation of their respective cellular targets for subsequent identification by mass spectrometry. When properly positioned, the tag does not significantly affect compound potency and membrane permeability, allowing for binding interactions with the tethered compound (probe) to be established within intact cells under physiological conditions. Subsequent UV-induced covalent photo-cross-linking "freezes" the interactions between the probe and its cellular targets and prevents their dissociation upon cell lysis. Targets cross-linked to the capture tag are then efficiently enriched through covalent capture onto HaloTag coated beads and subsequent selective chemical release from the solid support. The tag's built-in capability for selective enrichment eliminates the need for ligation of a capture tag, thereby simplifying the workflow and reducing variability introduced through additional operational steps. At the same time, the capacity for adequate cross-linking without structural optimization permits modular assembly of photoreactive chloroalkane probes, which reduces the burden of customized chemistry. Using three model compounds, we demonstrate the capability of this approach to identify known and novel cellular targets, including those with low affinity and/or low abundance as well as membrane targets with several transmembrane domains.

Interfacial Photo-Cross-Linking: Simple but Powerful Approach for Fabricating Capsule Polymer Particles with Tunable pH-Responsive Controlled Release Capability.

Herein, we describe capsule polymer particles with precisely controlled pH-responsive release properties prepared directly via the interfacial photo-cross-linking of spherical poly(2-diethylaminoethyl methacrylate-co-2-cinnamoylethyl methacrylate) (P(DEAEMA-CEMA)) particles. In the interfacial photo-cross-linking, photoreactive cinnamoyl groups in the polymer particles were cross-linked via [2π + 2π] cycloaddition reactions at the polymer/water interface, showing that the shell-cross-linked hollow polymer particles can be directly prepared from spherical polymer particles. The approach has fascinating advantages such as using minimal components, simplicity, and not requiring sacrificial template particles and toxic solvents. The following important observations are made: (I) encapsulated materials were stably retained in the capsule particles under neutral pH conditions; (II) encapsulated materials were released from the capsule particles under acidic pH conditions; (III) the release kinetics of encapsulated materials were controlled by the pH conditions; i.e., immediate and sustained release was achieved by varying the acidity of the aqueous media; (IV) the photoirradiation time did not significantly affect the release kinetics under different pH conditions; and (V) the pH-responsive release properties were regulated by changing the polymer composition in P(DEAEMA-CEMA). Furthermore, by exploiting the pH-responsiveness, capsule particles are successfully obtained via an all-aqueous process from spherical polymer particles. The advantages of the all-aqueous encapsulation process allowed the water-soluble biomacromolecules such as DNA and saccharides to be successfully encapsulated in the P(DEAEMA-CEMA) hollow particles. With this simple interfacial photo-cross-linking strategy, we envision the ready synthesis of sophisticated particulate materials for broad application in advanced research fields.

A Bifunctional NAD+ for Profiling Poly-ADP-Ribosylation-Dependent Interacting Proteins.

Protein poly-ADP-ribosylation (PARylation) is a heterogeneous and dynamic post-translational modification regulated by various writers, readers, and erasers. It participates in a variety of biological events and is involved in many human diseases. Currently, tools and technologies have yet to be developed for unambiguously defining readers and erasers of individual PARylated proteins or cognate PARylated proteins for known readers and erasers. Here, we report the generation of a bifunctional nicotinamide adenine dinucleotide (NAD+) characterized by diazirine-modified adenine and clickable ribose. By serving as an excellent substrate for poly-ADP-ribose polymerase 1 (PARP1)-catalyzed PARylation, the generated bifunctional NAD+ enables photo-cross-linking and enrichment of PARylation-dependent interacting proteins for proteomic identification. This bifunctional NAD+ provides an important tool for mapping cellular interaction networks centered on protein PARylation, which are essential for elucidating the roles of PARylation-based signals or activities in physiological and pathophysiological processes.

Thermally reversible and irreversible interstrand photocrosslinking of 5-chloro-2'-deoxy-4-thiouridine modified DNA oligonucleotides.

We describe highly efficient interstrand photocrosslinking of a DNA duplex containing 5-chloro-2'-deoxy-4-thiouridine (ClSdU) in one strand, proceeding via a two-step photochemical cascade, involving the formation of a thermally reversible crosslink between ClSdU and thymidine in the target strand and its subsequent conversion to a thermally stable fluorescent crosslink. These results show that ClSdU has great potential to be a valuable DNA photo-crosslinking reagent for chemical biology applications.

Photoinduced cross-linking of formulation buffer amino acids to monoclonal antibodies.

The correct choice of formulation buffer is a critical aspect of drug development and is chosen primarily to improve the stability of a protein therapeutic and protect against degradation. Amino acids are frequently incorporated into formulation buffers. In this study we have identified and characterized light induced cross-links between the side chain of histidine residues in an IgG4 monoclonal antibody and different amino acids commonly used in formulation buffers. These reactions have the potential to impact the overall product quality of the drug. The structure of each cross-link identified was elucidated using high performance liquid chromatography (HPLC) hyphenated to tandem mass spectrometry (MS/MS) with higher energy collisional dissociation (HCD). Furthermore, we speculate on the role of amino acids in formulation buffers and their influence on mAb stability. We theorize that whilst the adduction of formulation buffer amino acids could have a negative impact on product quality, it may protect against other pathways of photo-degradation.

Chemical Diversification of Simple Synthetic Antibodies.

Antibodies possess properties that make them valuable as therapeutics, diagnostics, and basic research tools. However, antibody chemical reactivity and covalent antigen binding are constrained, or even prevented, by the narrow range of chemistries encoded in canonical amino acids. In this work, we investigate strategies for leveraging an expanded range of chemical functionality using yeast displayed antibodies containing noncanonical amino acids (ncAAs) in or near antibody complementarity determining regions (CDRs). To enable systematic characterization of the effects of ncAA incorporation on antibody function, we first investigated whether diversification of a single antibody loop would support the isolation of binding clones against immunoglobulins from three species. We constructed and screened a billion-member library containing canonical amino acid diversity and loop length diversity only within the third complementarity determining region of the heavy chain (CDR-H3). Isolated clones exhibited moderate affinities (double- to triple-digit nanomolar affinities) and, in several cases, single-species specificity, confirming that antibody specificity can be mediated by a single CDR. This constrained diversity enabled the utilization of additional CDRs for the installation of chemically reactive and photo-cross-linkable ncAAs. Binding studies of ncAA-substituted antibodies revealed that ncAA incorporation is reasonably well tolerated, with observed changes in affinity occurring as a function of ncAA side chain identity, substitution site, and the ncAA incorporation machinery used. Multiple azide-containing ncAAs supported copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) without the abrogation of binding function. Similarly, several alkyne substitutions facilitated CuAAC without the apparent disruption of binding. Finally, antibodies substituted with a photo-cross-linkable ncAA were evaluated for ultraviolet-mediated cross-linking on the yeast surface. Competition-based assays revealed position-dependent covalent linkages, strongly suggesting successful cross-linking. Key findings regarding CuAAC reactions and photo-cross-linking on the yeast surface were confirmed using soluble forms of ncAA-substituted clones. The consistency of findings on the yeast surface and in solution suggest that chemical diversification can be incorporated into yeast display screening approaches. Taken together, our results highlight the power of integrating the use of yeast display and ncAAs in search of proteins with "chemically augmented" binding functions. This includes strategies for systematically introducing small molecule functionality within binding protein structures and evaluating protein-based covalent target binding. The efficient preparation and chemical diversification of antibodies on the yeast surface open up new possibilities for discovering "drug-like" protein leads in high throughput.

Retained Functionality of Atherosclerotic Human Arteries Following Photoactivated Linking of the Extracellular Matrix by Natural Vascular Scaffolding Treatment.

In this study, we investigated natural vascular scaffolding (NVS) treatment on vascular functionality using freshly isolated human popliteal arteries in vitro. Arteries were exposed to intraluminal NVS treatment consisting of a compound (4 amino-1,8-naphthalimide) photoactivated by a 450-nm light-emitting light fiber placed inside the artery. This procedure results in covalent linking between the extracellular matrix proteins to achieve a larger vessel diameter post-angioplasty and minimizing elastic recoil. Immediately following NVS treatment, rings were cut from the treated arteries and mounted in organ baths for contractility testing in response to U46619 and sodium nitroprusside. We also investigated the effect of NVS treatment on IL-6 cytokine release from vascular rings following a 4-h organoculture post-NVS treatment. Based on our results, we conclude that exposure of the vessels to NVS treatment does not adversely affect the contractile responsiveness of the vascular smooth muscle and exerts no pro-inflammatory effect. Graphical abstract.

Synthesis of an UV-Curable Divinyl-Fumarate Poly-ε-Caprolactone for Stereolithography Applications.

The limited number of commercially available photocrosslinkable resins for stereolithography has often been considered the main limitation of this technique. In this manuscript, a photocrosslinkable poly-ε-caprolactone (PCL) has been synthesized by a two-step method starting from ring opening polymerization (ROP) of ε-caprolactone. Hydroxyethyl vinyl ether (HEVE) has been used both as the initiator of ROP and as photo-curable functional group to obtain a vinyl poly-ε-caprolactone (VPCL). The following reaction of VPCL with fumaryl chloride (FuCl) results in a divinyl-fumarate polycaprolactone (VPCLF). Moreover, a catalyst based on Al, instead of the most popular Tin(II) 2-ethylhexanoate, has been employed to reduce the cytotoxicity of the material. VPCLF has been successfully used, in combination with N-vinyl-pyrrolidone (NVP), to fabricate 3D porous scaffolds by micro-stereolithography (µ-SL) with mathematically defined architectures.

Identification of SUMO Binding Proteins Enriched after Covalent Photo-Cross-Linking.

Post-translational modification with the small ubiquitin-like modifier (SUMO) affects thousands of proteins in the human proteome and is implicated in numerous cellular processes. The main outcome of SUMO conjugation is a rewiring of protein-protein interactions through recognition of the modifier's surface by SUMO binding proteins. The SUMO-interacting motif (SIM) mediates binding to a groove on SUMO; however, the low affinity of this interaction and the poor conservation of SIM sequences complicates the isolation and identification of SIM proteins. To address these challenges, we have designed and biochemically characterized monomeric and multimeric SUMO-2 probes with a genetically encoded photo-cross-linker positioned next to the SIM binding groove. Following photoinduced covalent capture, even weak SUMO binders are not washed away during the enrichment procedure, and very stringent washing conditions can be applied to remove nonspecifically binding proteins. A total of 329 proteins were isolated from nuclear HeLa cell extracts and identified using mass spectrometry. We found the molecular design of our probes was corroborated by the presence of many established SUMO interacting proteins and the high percentage (>90%) of hits containing a potential SIM sequence, as predicted by bioinformatic analyses. Notably, 266 of the 329 proteins have not been previously reported as SUMO binders using traditional noncovalent enrichment procedures. We confirmed SUMO binding with purified proteins and mapped the position of the covalent cross-links for selected cases. We postulate a new SIM in MRE11, involved in DNA repair. The identified SUMO binding candidates will help to reveal the complex SUMO-mediated protein network.

In situ photocrosslinkable formulation of nanocomposites based on multi-walled carbon nanotubes and formononetin for potential application in spinal cord injury treatment.

Carbon nanotubes (CN) have been studied to treat spinal cord injuries because of its electrical properties and nanometric dimensions. This work aims to develop a photopolymerizable hydrogel containing CN functionalized with an anti-inflammatory molecule to be used in situ on spinal cord injuries. The CN functionalization step was done using the drug (formononetin). The nanocomposites were characterized by morphological analysis, FTIR, Raman Spectroscopy, thermal analysis and cytotoxicity assays (MTT and HET-CAM). The nanocomposites were incorporated into gelatin methacryloyl hydrogel and exposed to UV light for photopolymerization. The volume of the formulation and the UV exposition time were also analyzed. The CN characterization showed that formononetin acted as a functionalization agent. The functionalized CN showed safe characteristics and can be incorporated in photocrosslinkable formulation. The UV exposition time for the formulation photopolymerization was compatible with the cell viability and also occurred in the injury site.

Biometals as conformational modulators of α-synuclein photochemical crosslinking.

Metal dyshomeostasis has long been linked to Parkinson's disease (PD), and the amyloidogenic protein α-synuclein (αS) is universally recognized as a key player in PD pathology. Structural consequences upon coordination of copper and iron to αS have gained attention due to significant dyshomeostasis of both metals in the PD brain. Protein-metal association can navigate protein folding in distinctive pathways based on the identity of the bio-metal in question. In this work, we employed photo-chemical crosslinking of unmodified proteins (PICUP) to evaluate these potential metal ion-induced structural alterations in the folding dynamics of N-terminally acetylated αS (NAcαS) following metal coordination. Through fluorescence analysis and immunoblotting analyses following photoirradiation, we discovered that coordination of iron obstructs copper-promoted crosslinking. The absence of intra-molecular crosslinking upon iron association further supports its C-terminal coordination site and suggests a potential role for iron in mitigating nearby post-translational modification of tyrosine residues. Decreased fluorescence emission upon synergistic coordination of both copper and iron highlighted that although copper acts as a conformational promotor of NAcαS crosslinking, iron inhibits analogous conformational changes within the protein. The metal coordination preferences of NAcαS suggest that both competitive binding sites as well as dual metal coordination contribute to the changes in folding dynamics, unveiling unique structural orientations for NAcαS that have a direct and measureable influence on photoinitiated dityrosine crosslinks. Moreover, our findings have physiological implications in that iron overload, as is associated with PD-insulted brain tissue, may serve as a conformational block of copper-promoted protein oxidation.

Evaluating Calmodulin-Protein Interactions by Rapid Photoactivated Cross-Linking in Live Cells Metabolically Labeled with Photo-Methionine.

This work addresses the question of how the Ca2+ sensor protein calmodulin shapes cellular responses to Ca2+ signals. Proteins interacting with affinity tagged calmodulin were captured by rapid (t1/2 ≈ 7 s) photoactivated cross-linking under basal conditions, after brief removal of extracellular Ca2+ and during a cytosolic [Ca2+] transient in cells metabolically labeled with a photoreactive methionine analog. Tagged adducts were stringently enriched, and captured proteins were identified and quantified by LC-MS/MS. A set of 489 proteins including 27 known calmodulin interactors was derived. A threshold for fractional capture was applied to define a high specificity group of 170 proteins, including 22 known interactors, and a low specificity group of 319 proteins. Capture of ∼60% of the high specificity group was affected by manipulations of Ca2+, compared with ∼20% of the low specificity group. This suggests that the former is likely to contain novel interactors of physiological significance. The capture of 29 proteins, nearly all high specificity, was decreased by the removal of extracellular Ca2+, although this does not affect cytosolic [Ca2+]. Capture of half of these was unaffected by the cytosolic [Ca2+] transient, consistent with high local [Ca2+]. These proteins are hypothesized to reside in or near microdomains of high [Ca2+] supported by the Ca2+ influx.

Quantitative Photo-crosslinking Mass Spectrometry Revealing Protein Structure Response to Environmental Changes.

Protein structures respond to changes in their chemical and physical environment. However, studying such conformational changes is notoriously difficult, as many structural biology techniques are also affected by these parameters. Here, the use of photo-crosslinking, coupled with quantitative crosslinking mass spectrometry (QCLMS), offers an opportunity, since the reactivity of photo-crosslinkers is unaffected by changes in environmental parameters. In this study, we introduce a workflow combining photo-crosslinking using sulfosuccinimidyl 4,4'-azipentanoate (sulfo-SDA) with our recently developed data-independent acquisition (DIA)-QCLMS. This novel photo-DIA-QCLMS approach is then used to quantify pH-dependent conformational changes in human serum albumin (HSA) and cytochrome C by monitoring crosslink abundances as a function of pH. Both proteins show pH-dependent conformational changes resulting in acidic and alkaline transitions. 93% and 95% of unique residue pairs (URP) were quantifiable across triplicates for HSA and cytochrome C, respectively. Abundance changes of URPs and hence conformational changes of both proteins were visualized using hierarchical clustering. For HSA we distinguished the N-F and the N-B form from the native conformation. In addition, we observed for cytochrome C acidic and basic conformations. In conclusion, our photo-DIA-QCLMS approach distinguished pH-dependent conformers of both proteins.

Benzophenone Photoreactivity in a Lipid Bilayer To Probe Peptide/Membrane Interactions: Simple System, Complex Information.

Affinity photo-cross-linking coupled to mass spectrometry, using benzophenone (Bzp)-functionalized peptides, was used to study the noncovalent interactions of cell-penetrating peptides and lipid membranes. Using biomimetic lipid vesicles composed of saturated and unsaturated negatively charged lipids, DMPG (14:0), DPPG (16:0), DOPG (18:1 cis Δ9), 18:1 (trans Δ9) PG, and DLoPG (18:2 cis Δ9, 12), allowed observation of all the classical and less common reactivities of Bzp described in the literature by direct MS analysis: C═C double bond formation on saturated fatty acids, covalent adducts formation via classical C-C bond, and Paternò-Büchi oxetane formation followed or not by fragmentation (retro-Paternò-Büchi) as well as photosensitization of unsaturated lipids leading to lipid dimers. All these reactions can occur concomitantly in a single complex biological system: a membrane-active peptide inserted within a phospholipid bilayer. We also detect oxidation species due to the presence of radical oxygen species. This work represents a noteworthy improvement for the characterization of interacting partners using Bzp photo-cross-linking, and it shows how to exploit in an original way the different reactivities of Bzp in the context of a lipid membrane. We propose an analytical workflow for the interpretation of MS spectra, giving access to information on the CPP/lipid interaction at a molecular level such as depth of insertion or membrane fluidity in the CPP vicinity. An application of this workflow illustrates the role of cholesterol in the CPP/lipids interaction.

Genetically Encoding Photocaged Quinone Methide to Multitarget Protein Residues Covalently in Vivo.

Genetically introducing covalent bonds into proteins in vivo with residue specificity is affording innovative ways for protein research and engineering, yet latent bioreactive unnatural amino acids (Uaas) genetically encoded to date react with one to few natural residues only, limiting the variety of proteins and the scope of applications amenable to this technology. Here we report the genetic encoding of (2 R)-2-amino-3-fluoro-3-(4-((2-nitrobenzyl)oxy) phenyl) propanoic acid (FnbY) in Escherichia coli and mammalian cells. Upon photoactivation, FnbY generated a reactive quinone methide (QM), which selectively reacted with nine natural amino acid residues placed in proximity in proteins directly in live cells. In addition to Cys, Lys, His, and Tyr, photoactivated FnbY also reacted with Trp, Met, Arg, Asn, and Gln, which are inaccessible with existing latent bioreactive Uaas. FnbY thus dramatically expanded the number of residues for covalent targeting in vivo. QM has longer half-life than the intermediates of conventional photo-cross-linking Uaas, and FnbY exhibited cross-linking efficiency higher than p-azido-phenylalanine. The photoactivatable and multitargeting reactivity of FnbY with selectivity toward nucleophilic residues will be valuable for addressing diverse proteins and broadening the scope of applications through exploiting covalent bonding in vivo for chemical biology, biotherapeutics, and protein engineering.

Reversible photo-cross-linking of the GCN4 peptide containing 3-cyanovinylcarbazole amino acid to double-stranded DNA.

Interaction analysis in vivo greatly promotes the analyses and understanding of biological functions. The interaction between DNA and peptides or proteins is very important in terms of readout and amplifying information from genomic DNA. In this study, we designed and synthesized a photo-cross-linkable amino acid, l-3-cyanovinlycarbazole amino acid (l-CNVA), to double-stranded DNA. Reversible photo-cross-linking between DNA and peptides containing CNVA, having 3-cyanovinylcarbazole moieties capable of photo-cross-linking to nucleic acids, was demonstrated. As a result, it was shown that the GCN4 peptide, containing CNVA, can be photo-cross-linked to DNA, and its adduct was photo-split into the original peptide and DNA with 312 nm-irradiation. This is the first report that reversibly manipulates photo-crosslinking between double stranded DNA and peptides. In addition, this reversible photo-cross-linking, using l-CNVA, is faster and with higher yield than that using diazirine and psoralen.

Visible Light Cross-Linking of Gelatin Hydrogels Offers an Enhanced Cell Microenvironment with Improved Light Penetration Depth.

In this study, the cyto-compatibility and cellular functionality of cell-laden gelatin-methacryloyl (Gel-MA) hydrogels fabricated using a set of photo-initiators which absorb in 400-450 nm of the visible light range are investigated. Gel-MA hydrogels cross-linked using ruthenium (Ru) and sodium persulfate (SPS), are characterized to have comparable physico-mechanical properties as Gel-MA gels photo-polymerized using more conventionally adopted photo-initiators, such as 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one (Irgacure 2959) and lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP). It is demonstrated that the Ru/SPS system has a less adverse effect on the viability and metabolic activity of human articular chondrocytes encapsulated in Gel-MA hydrogels for up to 35 days. Furthermore, cell-laden constructs cross-linked using the Ru/SPS system have significantly higher glycosaminoglycan content and re-differentiation capacity as compared to cells encapsulated using I2959 and LAP. Moreover, the Ru/SPS system offers significantly greater light penetration depth as compared to the I2959 system, allowing thick (10 mm) Gel-MA hydrogels to be fabricated with homogenous cross-linking density throughout the construct. These results demonstrate the considerable advantages of the Ru/SPS system over traditional UV polymerizing systems in terms of clinical relevance and practicability for applications such as cell encapsulation, biofabrication, and in situ cross-linking of injectable hydrogels.