Dataset Information

Photo Cross-Linking Probes Containing ?-N-Thioacyllysine and ?-N-Acyl-(?-aza)lysine Residues.

ABSTRACT: Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. This work describes the development of peptide activity/affinity-based probes for the discovery of proteins that recognize novel acyl-based PTMs on lysine residues in the proteome. The probes contain surrogates of ?-N-acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the experiments. In addition to the modified PTMs, the developed chemotypes were analyzed with respect to the effect of peptide sequence. The photo cross-linking conditions and subsequent functionalization of the covalent adducts were systematically optimized by applying fluorophore labeling and gel electrophoresis (in-gel fluorescence measurements). Finally, selected probes, containing the ?-N-glutaryllysine and ?-N-myristoyllysine analogues, were successfully applied for the enrichment of native, endogenous proteins from cell lysate, recapitulating the expected interactions of SIRT5 and SIRT2, respectively. Interestingly, the latter mentioned was able to pull down two different splice variants of SIRT2, which has not been achieved with a covalent probe before. Based on this elaborate proof-of-concept study, we expect that the technology will have broad future applications for pairing of novel PTMs with the proteins that target them in the cell.

SUBMITTER: Baek M

PROVIDER: S-EPMC7154546 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Publications

Photo Cross-Linking Probes Containing ϵ-N-Thioacyllysine and ϵ-N-Acyl-(δ-aza)lysine Residues.

Baek Michael M Martín-Gago Pablo P Laursen Jonas S JS Madsen Julie L H JLH Chakladar Saswati S Olsen Christian A CA

Chemistry (Weinheim an der Bergstrasse, Germany) 20200303 17

Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. This work describes the development of peptide activity/affinity-based probes for the discovery of proteins that recognize novel acyl-based PTMs on lysine residues in the proteome. The probes contain surrogates of ϵ-N-acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the ex ...[more]

PMID: 31922630

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Project description:Embedding nanomaterials into polymer hydrogels enables the design of functional materials with tailored chemical, mechanical, and optical properties. Nanocapsules that protect interior cargo and disperse readily through a polymeric matrix have drawn particular interest for their ability to integrate chemically incompatible systems and to further expand the parameter space for polymer nanocomposite hydrogels. The properties of polymer nanocomposite hydrogels depend on the material composition and processing route, which were explored systematically in this work. The gelation kinetics of network-forming polymer solutions with and without silica-coated nanocapsules bearing polyethylene glycol (PEG) surface ligands were investigated using in situ dynamic rheology measurements. Network-forming polymers comprised either 4-arm or 8-arm star PEG with terminal anthracene groups, which dimerize upon irradiation with ultraviolet (UV) light. The PEG-anthracene solutions exhibited rapid gel formation upon UV exposure (365 nm); gel formation was observed as a crossover from liquid-like to solid-like behavior during in situ small-amplitude oscillatory shear rheology. This crossover time was non-monotonic with polymer concentration. Far below the overlap concentration (c/c* ≪ 1), spatially separated PEG-anthracene molecules were subject to forming intramolecular loops over intermolecular cross-links, thereby slowing the gelation process. Near the polymer overlap concentration (c/c* ∼ 1), rapid gelation was attributed to the ideal proximity of anthracene end groups from neighboring polymer molecules. Above the overlap concentration (c/c* > 1), increased solution viscosities hindered molecular diffusion, thereby reducing the frequency of dimerization reactions. Adding nanocapsules to PEG-anthracene solutions resulted in faster gelation than nanocapsule-free PEG-anthracene solutions with equivalent effective polymer concentrations. The final elastic modulus of nanocomposite hydrogels increased with nanocapsule volume fraction, signifying synergistic mechanical reinforcement by nanocapsules despite not being cross-linked into the polymer network. Overall, these findings quantify the impact of nanocapsule addition on the gelation kinetics and mechanical properties of polymer nanocomposite hydrogels, which are promising materials for applications in optoelectronics, biotechnology, and additive manufacturing.

Dataset Information

Photo Cross-Linking Probes Containing ?-N-Thioacyllysine and ?-N-Acyl-(?-aza)lysine Residues.

Publications

Photo Cross-Linking Probes Containing ϵ-N-Thioacyllysine and ϵ-N-Acyl-(δ-aza)lysine Residues.

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