Metal-free functionalization of tyrosine residues in short peptides and study of the morphological alterations.

An efficient functionalization of tyrosine residues in phenolic regions is achieved under metal-free conditions. The strategy involves the conversion of a tyrosine residue to 4-amino phenylalanine or 4-amino-3-methoxy phenylalanine in short peptides through a controlled oxidative dearomatization. This transformation is achieved in one pot with good yields and excellent regioselectivity. Consequently, the self-assembly of the peptide compounds has been studied at the nanoscopic level before and after functionalization. The results suggest that the peptide derivatives comprising amide groups promote intermolecular H-bonding interactions and the difference in -OH and -NH2 functional groups is found to be responsible for the morphological changes. Morphological transitions from 1D nanowires to 2D nanosheets were observed during functional group modification.

Tyrosine nitration of glucagon impairs its function: Extending the role of heme in T2D pathogenesis.

New studies raise the possibility that the higher glucagon (GCG) level present in type 2 diabetes (T2D) is a compensatory mechanism to enhance ß-cell function, rather than induce dysregulated glucose homeostasis, due to an important role for GCG that acts directly within the pancreas on insulin secretion by intra-islet GCG signaling. However, in states of poorly controlled T2D, pancreatic α cell mass increases (overproduced GCG) in response to insufficient insulin secretion, indicating decreased local GCG activity. The reason for this decrease is not clear. Recent evidence has uncovered a new role of heme in cellular signal transduction, and its mechanism involves reversible binding of heme to proteins. Considering that protein tyrosine nitration in diabetic islets increases and glucose-stimulated insulin secretion (GSIS) decreases, we speculated that heme modulates GSIS by transient interaction with GCG and catalyzing its tyrosine nitration, and the tyrosine nitration may impair GCG activity, leading to loss of intra-islet GCG signaling and markedly impaired insulin secretion. Data presented here elucidate a novel role for heme in disrupting local GCG signaling in diabetes. Heme bound to GCG and induced GCG tyrosine nitration. Two tyrosine residues in GCG were both sensitive to the nitrating species. Further, GCG was also demonstrated to be a preferred target peptide for tyrosine nitration by co-incubation with BSA. Tyrosine nitration impaired GCG stimulated cAMP-dependent signaling in islet ß cells and decreased insulin release. Our results provided a new role of heme for impaired GSIS in the pathological process of diabetes.

Non-fluorescent transient states of tyrosine as a basis for label-free protein conformation and interaction studies.

The amino acids tryptophan, tyrosine, and phenylalanine have been extensively used for different label-free protein studies, based on the intensity, lifetime, wavelength and/or polarization of their emitted fluorescence. Similar to most fluorescent organic molecules, these amino acids can undergo transitions into dark meta-stable states, such as triplet and photo-radical states. On the one hand, these transitions limit the fluorescence signal, but they are also highly environment-sensitive and can offer an additional set of parameters, reflecting interactions, folding states, and immediate environments around the proteins. In this work, by analyzing the average intensity of tyrosine emission under different excitation modulations with the transient state monitoring (TRAST) technique, we explored the photo physics of tyrosine as a basis for such environment-sensitive readouts. From how the dark state transitions of tyrosine varied with excitation intensity and solvent conditions we first established a photophysical model for tyrosine. Next, we studied Calmodulin (containing two tyrosines), and how its conformation is changed upon calcium binding. From these TRAST experiments, performed with 280 nm time-modulated excitation, we show that tyrosine dark state transitions clearly change with the calmodulin conformation, and may thus represent a useful source of information for (label-free) analyses of protein conformations and interactions.

Solution-Phase Late-Stage Chemoselective Photocatalytic Removal of Sulfonyl and Phenacyl Groups in Peptides.

Herein, BPC catalyzed visible-light-triggered target-specific late-stage solution phase desulfonylation from tryptophan in oligopeptides is portrayed by overcoming the isolation issue up to octamers. This robust and mild method is highly predictable and chemoselective, tolerating myriad of functional groups in aza-heteroaromatics and peptides. Interestingly, reductive desulfonylation is also amenable to biologically significant reactive histidine and tyrosine side chains, signifying the versatility of the strategy. Additional efficacy of BPC is demonstrated by solution phase phenacyl deprotection from C-terminal in peptides. Furthermore, excellent catalyst loading of 0.5 mol% and recyclability demonstrate the practical utility and applicability of this strategy.

Self-Assembled Materials Based on Fully Aromatic Peptides: The Impact of Tryptophan, Tyrosine, and Dopa Residues.

Peptides are able to self-organize in structural elements including cross-ß structures. Taking advantage of this tendency, in the last decades, peptides have been scrutinized as molecular elements for the development of multivalent supramolecular architectures. In this context, different classes of peptides, also with completely aromatic sequences, were proposed. Our previous studies highlighted that the (FY)3 peptide, which alternates hydrophobic phenylalanine and more hydrophilic tyrosine residues, is able to self-assemble, thanks to the formation of both polar and apolar interfaces. It was observed that the replacement of Phe and Tyr residues with other noncoded aromatic amino acids like 2-naphthylalanine (Nal) and Dopa affects the interactions among peptides with consequences on the supramolecular organization. Herein, we have investigated the self-assembling behavior of two novel (FY)3 analogues with Trp and Dopa residues in place of the Phe and Tyr ones, respectively. Additionally, PEGylation of the N-terminus was analyzed too. The supramolecular organization, morphology, and capability to gel were evaluated using complementary techniques, including fluorescence, Fourier transform infrared spectroscopy, and scanning electron microscopy. Structural periodicities along and perpendicular to the fiber axis were detected by grazing incidence wide-angle X-ray scattering. Finally, molecular dynamics studies provided interesting insights into the atomic structure of the cross-ß that constitutes the basic motif of the assemblies formed by these novel peptide systems.

Structure-function relationship study for sulfated protein therapeutics using hydrophobic interaction chromatography and mass spectrometry.

Protein tyrosine sulfation is a post-translational modification (PTM) that is rarely reported in recombinant therapeutic proteins. However, when sulfation does occur, the additional negative charge from the modification can influence intermolecular interactions and antigen-binding activity, making it a critical quality attribute that necessitates stringent control. In this study, we developed a unique hydrophobic interaction chromatography (HIC) method for the separation and quantification of a therapeutic bispecific antibody with varying degrees of sulfation. Despite the increased surface hydrophilicity of sulfated species, the HIC method provides enhanced retention. Baseline resolution was attained based on the degree of sulfation, independent of other PTMs such as C-terminal amidation and forced deamidation. Further structure-function relationship studies of enriched sulfated bispecific antibody species were conducted using mass spectrometry and fluorescence-linked immunosorbent assay (FLISA). These studies revealed that the tyrosine sulfation modification, which occurs in the complementarity-determining region (CDR), is a critical quality attribute and can adversely impact the antibody's binding to its cognate antigen. The evaluation of sulfation assay using HIC method confirmed it is an effective means for controlling this critical quality attribute.

Naked-eye rapid recognition of tyrosine enantiomers using silver triangular nanoplates as colorimetric probe.

Recognizing and quantifying enantiomers of chiral molecule is of great importance in chemical, biological and pharmaceutical fields. Herein, we presented one simple-yet-efficient method of sensing tyrosine (Tyr) enantiomers. In this sensing, silver triangular nanoplates (AgTNPs) were used as colorimetric probes. L-Tyr quickly induced the color of AgTNPs solution to change from dark blue to light gray, whereas D-Tyr induced no change of the AgTNPs solution color. The obvious color change enables the naked eye to recognize Tyr enantiomer. The visual method was used to detect the enantiometric excess value of L-Tyr in the whole range (-100 % ∼ 100 %). This chiral sensing can be finished within 5 min using one simple ultraviolet-visible spectrometer or naked eye. Furthermore, the mechanism of this chiral sensing was explored. It was confirmed that this chiral sensing was based on AgTNPs' intrinsic chirality. This chiral sensing is rapid, simple, and low-cost, and has great potential for chiral determination of Tyr.

Highly efficient site-specific protein modification using tyrosinase from Streptomyces avermitilis: Structural insight.

Tyrosinase-mediated protein conjugation has recently drawn attention as a site-specific protein modification tool under mild conditions. However, the tyrosinases reported to date act only on extremely exposed tyrosine residues, which limits where the target tyrosine can be located. Herein, we report a tyrosinase from Streptomyces avermitilis (SaTYR), that exhibits a much higher activity against tyrosine residues on the protein surface than other tyrosinases. We determined the crystal structure of SaTYR and revealed that the enzyme has a relatively flat and shallow substrate-binding pocket to accommodate a protein substrate. We demonstrated SaTYR-mediated fluorescence dye tagging and PEGylation of a surface tyrosine residue that was unreacted by other tyrosinases with an approximately 95.2 % conjugation yield in 1 h. We also present a structural rationale that considers the steric hindrance from adjacent residues and surrounding structures along with the extent of solvent exposure of residues, as necessary when determining the optimal positions for introducing target tyrosine residues in SaTYR-mediated protein modification. The study demonstrated that the novel tyrosinase, SaTYR, extends the scope of tyrosinase-mediated protein modification, and we propose that site-specific tyrosine conjugation using SaTYR is a promising strategy for protein bioconjugation in various applications.

Molecular Recognition of Tyrosine-Containing Polypeptides with Pseudopeptidic Cages Unraveled by Fluorescence and NMR Spectroscopies.

The molecular recognition of Tyr-containing peptide copolymers with pseudopeptidic cages has been studied using a combination of fluorescence and NMR spectroscopies. Fluorescence titrations rendered a reasonable estimation of the affinities, despite the presence of dynamic quenching masking the unambiguous detection of the supramolecular complexes. Regarding NMR, the effect of polypeptide (PP) binding on relaxation and diffusion parameters of the cages is much more reliable than the corresponding chemical shift perturbations. To that, purification of the commercial PPs is mandatory to obtain biopolymers with lower polydispersity. Thus, the relaxation/diffusion-filtered 1H spectra of the cages in the absence vs presence of the PPs represent a suitable setup for the fast detection of the noncovalent interactions. Additional key intermolecular NOE cross-peaks supported by molecular models allow the proposal of a structure of the supramolecular species, stabilized by the Tyr encapsulation within the cage cavity and additional attractive polar interactions between the side chains of cage and PP, thus defining a binding epitope with a potential for implementing sequence selectivity. Accordingly, the cages bearing positive/negative residues prefer to bind the peptides having complementary negative/positive side chains close to the target Tyr, suggesting an electrostatic contribution to the interaction. Overall, our results show that both techniques represent a powerful and complementary combination for studying cage-to-PP molecular recognition processes.

A computational insight on the aromatic amino acids conjugation with [Cp*Rh(H2O)3]2+ by using the meta-dynamics/FMO3 approach.

CONTEXT: Rh(III) complexes demonstrated to exert promising pharmacological effects with potential applications as anti-cancer, anti-bacterial, and antimicrobial agents. One important Rh(III)-ligand is the pentamethylcyclopentadienyl (Cp*) group forming in water the [Cp*Rh(H2O)3]2+ complex. Among of its attractive chemical properties is the ability to react specifically with Tyr amino acid side chain of G-protein-coupled receptor (GPCR) peptides by means of highly chemoselective bioconjugation reaction, at room temperature and at pH 5-6. In this computational work, in order to deepen the mechanism of this chemoselective conjugation, we study the ligand exchange reaction between [Cp*Rh(H2O)3]2+ and three small molecules, namely p-cresol, 3-methylimidazole, and toluene, selected as mimetic of aromatic side chains of tyrosine (Tyr), tryptophan (Trp) and phenylalanine (Phe), respectively. Our outcomes suggest that the high selectivity for Tyr side chain might be related to OH group able to affect both thermodynamic and kinetic of ligand exchange reaction, due to its ability to act as both H bond acceptor and donor. These mechanistic aspects can be used to design new metal drugs containing the [Cp*Rh]2+ scaffold targeting specifically Tyr residues involved in biological/pathological processes such as phosphorylation by means of Tyr-kinase enzyme and protein-protein interactions. METHODS: The geometry of three encounter complexes and product adducts were optimized at the B3LYP//CPCM/ωB97X-D level of theory, adopting the 6-311+G(d,p) basis set for all non-metal atoms and the LANL2DZ pseudopotential for the Rh atom. Meta-dynamics RMSD (MTD(RMSD)) calculations at GFN2-xTB level of theory were performed in NVT conditions at 298.15 K to investigate the bioconjugation reactions (simulation time: 100 ps; integration step 2.0; implicit solvent model: GBSA). The MTD(RMSD) simulation was performed in two replicates for each encounter complex. Final representative subsets of 100 structures for each run were gained with a sampling rate of 1 ps and analyzed by performing single point calculations using the FMO3 method at RI-MP2/6-311G//PCM[1] level of theory, adopting the MCP-TZP core potential for Rh atom.

Theoretical Insights into the Generation Mechanism of the Tyr122 Radical Catalyzed by Intermediate X in Class Ia Ribonucleotide Reductase.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides in all organisms. There is an ∼35 Å long-range electron-hole transfer pathway during the catalytic process of class Ia RNR, which can be described as Tyr122ß â†” [Trp48ß]? ↔ Tyr356ß â†” Tyr731α ↔ Tyr730α ↔ Cys439α. The formation of the Y122• radical initiates this long-range radical transfer process. However, the generation mechanism of Y122• is not yet clear due to confusion over the intermediate X structures. Based on the two reported X structures, we examined the possible mechanisms of Y122• generation by density functional theory (DFT) calculations. Our examinations revealed that the generation of the Y122• radical from the two different core structures of X was via a similar two-step reaction, with the first step of proton transfer for the formation of the proton receptor of Y122 and the second step of a proton-coupled long-range electron transfer reaction with the proton transfer from the Y122 hydroxyl group to the terminal hydroxide ligand of Fe1III and simultaneously electron transfer from the side chain of Y122 to Fe2IV. These findings provide an insight into the formation mechanism of Y122• catalyzed by the double-iron center of the ß subunit of class Ia RNR.

Identification of tyrosine sulfation in the variable region of a bispecific antibody and its effect on stability and biological activity.

Despite tyrosine sulfation being a relatively common post-translational modification (PTM) on the secreted proteins of higher eukaryotic organisms, there have been surprisingly few reports of this modification occurring in recombinant monoclonal antibodies (mAbs) expressed by mammalian cell lines and even less information regarding its potential impact on mAb efficacy and stability. This discrepancy is likely due to the extreme lability of this modification using many of the mass spectrometry methods typically used within the biopharmaceutical industry for PTM identification, as well as the possible misidentification as phosphorylation. Here, we identified sulfation on a single tyrosine residue located within the identical variable region sequence of a 2 + 1 bispecific mAbs heavy and heavy-heavy chains using a multi-enzymatic approach in combination with mass spectrometry analysis and examined its impact on binding, efficacy, and physical stability. Unlike previous reports, we found that tyrosine sulfation modestly decreased the mAb cell binding and T cell-mediated killing, primarily by increasing the rate of antigen disassociation as determined from surface plasmon resonance-binding experiments. We also found that, while this acidic modification had no significant impact on the mAb thermal stability, sulfation did modestly increase its rate of aggregation, presumably by lowering the mAb's colloidal stability as indicated by polyethylene glycol induced liquid-liquid phase separation experiments.

Exploiting Chemical Protein Synthesis to Study the Role of Tyrosine Sulfation on Anticoagulants from Hematophagous Organisms.

Tyrosine sulfation is a post-translational modification (PTM) that modulates function by mediating key protein-protein interactions. One of the early proteins shown to possess this PTM was hirudin, produced in the salivary glands of the medicinal leech Hirudo medicinalis, whereby tyrosine sulfation led to a ∼10-fold improvement in α-thrombin inhibitory activity. Outside of this pioneering discovery, the involvement of tyrosine sulfation in modulating the activity of salivary proteins from other hematophagous organisms was unknown. We hypothesized that the intrinsic instability of the tyrosine sulfate functionality, particularly under the acidic conditions used to isolate and analyze peptides and proteins, has led to poor detection during the isolation and/or expression of these molecules.Herein, we summarize our efforts to interrogate the functional role of tyrosine sulfation in the thrombin inhibitory and anticoagulant activity of salivary peptides and proteins from a range of different blood feeding organisms, including leeches, ticks, mosquitoes, and flies. Specifically, we have harnessed synthetic chemistry to efficiently generate homogeneously sulfated peptides and proteins for detailed structure-function studies both in vitro and in vivo.Our studies began with the leech protein hirudin P6 (from Hirudinaria manillensis), which is both sulfated on tyrosine and O-glycosylated at a nearby threonine residue. Synthetically, this was achieved through solid-phase peptide synthesis (SPPS) with a late-stage on-resin sulfation, followed by native chemical ligation and a folding step to generate six differentially modified variants of hirudin P6 to assess the functional interplay between O-glycosylation and tyrosine sulfation. A one-pot, kinetically controlled ligation of three peptide fragments was used to assemble homogeneously sulfoforms of madanin-1 and chimadanin from the tick Haemaphysalis longicornis. Dual tyrosine sulfation at two distinct sites was shown to increase the thrombin inhibitory activity by up to 3 orders of magnitude through a novel interaction with exosite II of thrombin. The diselenide-selenoester ligation developed by our lab provided us with a means to rapidly assemble a library of different sulfated tick anticoagulant proteins: the andersonins, hyalomins, madanin-like proteins, and hemeathrins, thus enabling the generation of key structure-activity data on this family of proteins. We have also confirmed the presence of tyrosine sulfation in the anticoagulant proteins of Anopheles mosquitoes (anophelins) and the Tsetse fly (TTI) via insect expression and mass spectrometric analysis. These molecules were subsequently synthesized and assessed for thrombin inhibitory and anticoagulant activity. Activity was significantly improved by the addition of tyrosine sulfate modifications and led to molecules with potent antithrombotic activity in an in vivo murine thrombosis model.The Account concludes with our most recent work on the design of trivalent hybrids that tandemly occupy the active site and both exosites (I and II) of α-thrombin, with a TTI-anophelin hybrid (Ki = 20 fM against α-thrombin) being one of the most potent protease inhibitors and anticoagulants ever generated. Taken together, this Account highlights the importance of the tyrosine sulfate post-translational modification within salivary proteins from blood feeding organisms for enhancing anticoagulant activity. This work lays the foundation for exploiting native or engineered variants as therapeutic leads for thrombotic disorders in the future.

Free-Energy Landscape and Rate Estimation of the Aromatic Ring Flips in Basic Pancreatic Trypsin Inhibitors Using Metadynamics.

Aromatic side chains (phenylalanine and tyrosine) of a protein flip by 180° around the Cß-Cγ axis (χ2 dihedral of the side chain), producing two symmetry-equivalent states. The study of ring flip dynamics with nuclear magnetic resonance (NMR) experiments helps to understand local conformational fluctuations. Ring flips are categorized as slow (milliseconds and onward) or fast (nanoseconds to near milliseconds) based on timescales accessible to NMR experiments. In this study, we investigated the ability of the infrequent metadynamics approach to estimate the flip rate and discriminate between slow and fast ring flips for eight individual aromatic side chains (F4, Y10, Y21, F22, Y23, F33, Y35, and F45) of the basic pancreatic trypsin inhibitor. Well-tempered metadynamics simulations were performed to estimate the ring-flipping free-energy surfaces for all eight aromatic residues. The results indicate that χ2 as a standalone collective variable (CV) is not sufficient to obtain computationally consistent results. Inclusion of a complementary CV, such as χ1(Cα-Cß), solved the problem for most residues and enabled us to classify fast and slow ring flips. This indicates the importance of librational motions in ring flips. Multiple pathways and mechanisms were observed for residues F4, Y10, and F22. Recrossing events were observed for residues F22 and F33, indicating a possible role of friction effects in ring flipping. The results demonstrate the successful application of infrequent metadynamics to estimate ring flip rates and identify certain limitations of the approach.

Chemokine binding to PSGL-1 is controlled by O-glycosylation and tyrosine sulfation.

Protein glycosylation influences cellular recognition and regulates protein interactions, but how glycosylation functions alongside other common posttranslational modifications (PTMs), like tyrosine sulfation (sTyr), is unclear. We produced a library of 53 chemoenzymatically synthesized glycosulfopeptides representing N-terminal domains of human and murine P-selectin glycoprotein ligand-1 (PSGL-1), varying in sTyr and O-glycosylation (structure and site). Using these, we identified key roles of PSGL-1 O-glycosylation and sTyr in controlling interactions with specific chemokines. Results demonstrate that sTyr positively affects CCL19 and CCL21 binding to PSGL-1 N terminus, whereas O-glycan branching and sialylation reduced binding. For murine PSGL-1, interference between PTMs is greater, attributed to proximity between the two PTMs. Using fluorescence polarization, we found sTyr is a positive determinant for some chemokines. We showed that synthetic sulfopeptides are potent in decreasing chemotaxis of human dendritic cells toward CCL19 and CCL21. Our results provide new research avenues into the interplay of PTMs regulating leukocyte/chemokine interactions.

Ultraviolet Resonance Raman Spectra of Serum Albumins.

The ultraviolet resonance Raman (UVRR) spectra of the two proteins bovine serum albumin (BSA) and human serum albumin (HSA) in an aqueous solution are compared with the aim to distinguish between them based on their very similar amino acid composition and structure and to obtain signals from tryptophan that has only very few residues. Comparison of the protein spectra with solutions of tryptophan, tyrosine, and phenylalanine in comparative ratios as in the two proteins shows that at an excitation wavelength of 220 nm, the spectra are dominated by the strong resonant contribution from these three amino acids. While the strong enhancement of two and one single tryptophan residue in BSA and HSA, respectively, results in pronounced bands assigned to fundamental vibrations of tryptophan, its weaker overtones and combination bands do not play a major role in the spectral range above 1800 cm-1. There, the protein spectra clearly reveal the signals of overtones and combination bands of phenylalanine and tyrosine. Assignments of spectral features in the range of Raman shifts from 3800 to 5100 cm-1 to combinations comprising fundamentals and overtones of tyrosine were supported by spectra of amino acid mixtures that contain deuterated tyrosine. The information in the high-frequency region of the UVRR spectra could provide information that is complementary to near-infrared absorption spectroscopy of the proteins.

Improving the catalytic ability of a peptide-based artificial glycosidase through a tyrosine strategy.

Recently, peptide-based artificial enzymes have attracted growing interest due to the similar composition of peptide assemblies to natural enzymes. However, low catalytic activity and stability are still the main challenges for these enzyme-like catalysts. In this study, a tyrosine strategy was developed to construct a peptide-based artificial glycosidase through engaging the Tyr residue into the peptide sequence. We found that the appropriate substitution of Tyr produced an enhanced catalytic ability, because the proximal Tyr residue around catalytic sites acts as a nucleophile, playing an important role in the substrate orientation and proton transfer. Moreover, inspired by the biological function of dityrosine bonds in structural proteins, a photo-crosslinking reaction between the phenolic side chains of Tyr was also employed to form intermolecular covalent bonds in peptide assemblies, which further improved the activity and stability of the artificial glycosidase. This work provides a simple avenue for the designing and construction of efficient glycosidase-like catalysts based on a peptide-based material platform.

Tyrosine residues initiated photopolymerization in living organisms.

Towards intracellular engineering of living organisms, the development of new biocompatible polymerization system applicable for an intrinsically non-natural macromolecules synthesis for modulating living organism function/behavior is a key step. Herein, we find that the tyrosine residues in the cofactor-free proteins can be employed to mediate controlled radical polymerization under 405 nm light. A proton-coupled electron transfer (PCET) mechanism between the excited-state TyrOH* residue in proteins and the monomer or the chain transfer agent is confirmed. By using Tyr-containing proteins, a wide range of well-defined polymers are successfully generated. Especially, the developed photopolymerization system shows good biocompatibility, which can achieve in-situ extracellular polymerization from the surface of yeast cells for agglutination/anti-agglutination functional manipulation or intracellular polymerization inside yeast cells, respectively. Besides providing a universal aqueous photopolymerization system, this study should contribute a new way to generate various non-natural polymers in vitro or in vivo to engineer living organism functions and behaviours.

Improved spectral resolution of [13C,1H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive 13C-labelled precursors.

Cell-free protein synthesis using eCells allows production of amino acids from inexpensive 13C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amino acids is maintained in eCells. Judicious choice of 13C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display [13C,1H]-HSQC cross-peaks free of one-bond 13C-13C couplings. Selective 13C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers.

Photoradical-Mediated Catalyst-Independent Protein Cross-Link with Unusual Fluorescence Properties.

Photo-actively modified natural amino acids have served as lucrative probes for precise mapping of the dynamics, interaction networks, and turnover of cytosolic proteins both in vivo and ex vivo. In our attempts to extend the utility of photoreactive reporters to map the molecular characteristics of vital membrane proteins, we carried out site-selective incorporation of 7-fluoro-indole in the human mitochondrial outer membrane protein VDAC2 (voltage-dependent anion channel isoform 2), with the aim of generating Trp-Phe/Tyr cross-links. Prolonged irradiation at 282 nm provided us with a surprisingly unusual fluorophore that displayed sizably red-shifted excitation (λex-max =280 nm→360 nm) and emission (λem-max =330 nm→430 nm) spectra that was reversible with organic solvents. By measuring the kinetics of the photo-activated cross-linking with a library of hVDAC2 variants, we demonstrate that formation of this unusual fluorophore is kinetically retarded, independent of tryptophan, and is site-specific. Using other membrane (Tom40 and Sam50) and cytosolic (MscR and DNA Pol I) proteins, we additionally show that formation of this fluorophore is protein-independent. Our findings reveal the photoradical-mediated accumulation of reversible tyrosine cross-links, with unusual fluorescent properties. Our findings have immediate applications in protein biochemistry and UV-mediated protein aggregation and cellular damage, opening avenues for formulating therapeutics that prolong cell viability in humans.