Linchpins empower promiscuous electrophiles to enable site-selective modification of histidine and aspartic acid in proteins.
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ABSTRACT:
SUBMITTER: Rawale DG
PROVIDER: S-EPMC8133000 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Site-selective conjugation generally requires both (i) molecular engineering of the protein of interest to introduce a conjugation site at a defined location and (ii) a site-specific conjugation technology. Three N-terminal interferon α2-a (IFN) variants with truncated histidine tags were prepared and conjugation was examined using a bis-alkylation reagent, PEG(10kDa)-mono-sulfone 3. A histidine tag comprised of two histidines separated by a glycine (His2-tag) underwent PEGylation. Two more IFN variants were then prepared with the His2-tag engineered at different locations in IFN. Another IFN variant was prepared with the His-tag introduced in an α-helix, and required three contiguous histidines to ensure that two histidine residues in the correct conformation would be available for conjugation. Since histidine is a natural amino acid, routine methods of site-directed mutagenesis were used to generate the IFN variants from E. coli in soluble form at titres comparable to native IFN. PEGylation conversions ranged from 28-39%. A single step purification process gave essentially the pure PEG-IFN variant (>97% by RP-HPLC) in high recovery with isolated yields ranging from 21-33%. The level of retained bioactivity was strongly dependent on the site of PEG conjugation. The highest biological activity of 74% was retained for the PEG10-106(HGHG)-IFN variant which is unprecedented for a PEGylated IFN. The His2-tag at 106(HGHG)-IFN is engineered at the flexible loop most distant from IFN interaction with its dimeric receptor. The biological activity for the PEG10-5(HGH)-IFN variant was determined to be 17% which is comparable to other PEGylated IFN conjugates achieved at or near the N-terminus that have been previously described. The lowest retained activity (10%) was reported for PEG10-120(HHH)-IFN which was prepared as a negative control targeting a IFN site thought to be involved in receptor binding. The presence of two histidines as a His2-tag to generate a site-selective target for bis-alkylating PEGylation is a feasible approach for achieving site-selective PEGylation. The use of a His2-tag to strategically engineer a conjugation site in a protein location can result in maximising the retention of the biological activity following protein modification.
| S-EPMC6354831 | biostudies-literature
Project description:In chemical biology, azides are used to chemically manipulate target structures in a bioorthogonal manner for a plethora of applications ranging from target identification to the synthesis of homogeneously modified protein conjugates. While a variety of methods have been established to introduce the azido group into recombinant proteins, a method that directly converts specific amino groups in endogenous proteins is lacking. Here, we report the first biotin-tethered diazotransfer reagent DtBio and demonstrate that it selectively modifies the model proteins streptavidin and avidin and the membrane protein BioY on cell surface. The reagent converts amines in the proximity of the binding pocket to azides and leaves the remaining amino groups in streptavidin untouched. Reagents of this novel class will find use in target identification as well as the selective functionalization and bioorthogonal protection of proteins.
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Project description:Oxetanes are four-membered ring oxygen heterocycles that are advantageously used in medicinal chemistry as modulators of physicochemical properties of small molecules. Herein, we present a simple method for the incorporation of oxetanes into proteins through chemoselective alkylation of cysteine. We demonstrate a broad substrate scope by reacting proteins used as apoptotic markers and in drug formulation, and a therapeutic antibody with a series of 3-oxetane bromides, enabling the identification of novel handles (S-to-S/N rigid, non-aromatic, and soluble linker) and reactivity modes (temporary cysteine protecting group), while maintaining their intrinsic activity. The possibility to conjugate oxetane motifs into full-length proteins has potential to identify novel drug candidates as the next-generation of peptide/protein therapeutics with improved physicochemical and biological properties.
| S-EPMC5434895 | biostudies-literature
Project description: Not available
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Project description:Post-translational modifications (PTMs) are used by organisms to control protein structure and function after protein translation, but their study is complicated and their roles are not often well understood as PTMs are difficult to introduce onto proteins selectively. Designing reagents that are both good mimics of PTMs, but also only modify select amino acid residues in proteins is challenging. Frequently, both a chemical warhead and linker are used, creating a product that is a misrepresentation of the natural modification. We have previously shown that biotin-chloromethyl-triazole is an effective reagent for cysteine modification to give S-Lys derivatives where the triazole is a good mimic of natural lysine acylation. Here, we demonstrate both how the reactivity of the alkylating reagents can be increased and how the range of triazole PTM mimics can be expanded. These new iodomethyl-triazole reagents are able to modify a cysteine residue on a histone protein with excellent selectivity in 30 min to give PTM mimics of acylated lysine side-chains. Studies on the more complicated, folded protein SCP-2L showed promising reactivity, but also suggested the halomethyl-triazoles are potent alkylators of methionine residues.
| S-EPMC8471731 | biostudies-literature
Project description:Many biomedical fields rely on proteins that are selectively modified. These can be attached using reactive or catalytic moieties, but the position where these moieties are attached is often poorly controlled. We assessed how catalyst position affects the efficiency and selectivity of protein modification. For this, we anchored a template DNA strand to three different proteins, which were subsequently hybridized to DNA strands that contained catalysts at different positions. We found a strong correlation between the catalyst-to-protein distance and the efficiency of protein modification for acyl transfer catalysts, which operate via a covalently bound reactant intermediate. Additionally, we found that the catalyst's distance and orientation with respect to the protein surface, also influences its site-selectivity. A catalyst operating with unbound reactant intermediates showed only enhanced efficiency. Our results are rationalized using computational simulations, showing that one-point anchoring of the DNA construct leads to notable differences in the site of modification.
| S-EPMC9546015 | biostudies-literature
Project description:The reaction of meso-tetrakis(pentafluorophenyl)porpholactone with azomethine ylides and nitrones affords pyrrolidine-fused and isoxazolidine-fused dihydroporpholactones that display, respectively, isobacteriochlorin- and chlorin-type UV-Vis spectra. These reactions are site-selective, yielding, respectively, 17,18- or 12,13-dihydroporpholactones. The crystal and molecular features of pyrrolidine-fused and isoxazolidine-fused dihydroporpholactones were unveiled from single-crystal X-ray diffraction studies.
| S-EPMC7321334 | biostudies-literature
Project description:Experimental studies on protein dynamics at atomic resolution by NMR-spectroscopy in solution require isolated 1H-X spin pairs. This is the default scenario in standard 1H-15N backbone experiments. Side chain dynamic experiments, which allow to study specific local processes like proton-transfer, or tautomerization, require isolated 1H-13C sites which must be produced by site-selective 13C labeling. In the most general way this is achieved by using site-selectively 13C-enriched glucose as the carbon source in bacterial expression systems. Here we systematically investigate the use of site-selectively 13C-enriched ribose as a suitable precursor for 13C labeled histidines and tryptophans. The 13C incorporation in nearly all sites of all 20 amino acids was quantified and compared to glucose based labeling. In general the ribose approach results in more selective labeling. 1-13C ribose exclusively labels His ?2 and Trp ?1 in aromatic side chains and helps to resolve possible overlap problems. The incorporation yield is however only 37% in total and 72% compared to yields of 2-13C glucose. A combined approach of 1-13C ribose and 2-13C glucose maximizes 13C incorporation to 75% in total and 150% compared to 2-13C glucose only. Further histidine positions ?, ? and CO become significantly labeled at around 50% in total by 3-, 4- or 5-13C ribose. Interestingly backbone CO of Gly, Ala, Cys, Ser, Val, Phe and Tyr are labeled at 40-50% in total with 3-13C ribose, compared to 5% and below for 1-13C and 2-13C glucose. Using ribose instead of glucose as a source for site-selective 13C labeling enables a very selective labeling of certain positions and thereby expanding the toolbox for customized isotope labeling of amino-acids.
| S-EPMC5626788 | biostudies-literature
Project description:The synthesis of eukaryotic glycans - branched sugar oligomers attached to cell-surface proteins and lipids - is organized like a factory assembly line. Specific enzymes within successive compartments of the Golgi apparatus determine where new monomer building blocks are linked to the growing oligomer. These enzymes act promiscuously and stochastically, causing microheterogeneity (molecule-to-molecule variability) in the final oligomer products. However, this variability is tightly controlled: a given eukaryotic protein type is typically associated with a narrow, specific glycan oligomer profile. Here, we use ideas from the mathematical theory of self-assembly to enumerate the enzymatic causes of oligomer variability and show how to eliminate each cause. We rigorously demonstrate that cells can specifically synthesize a larger repertoire of glycan oligomers by partitioning promiscuous enzymes across multiple Golgi compartments. This places limits on biomolecular assembly: glycan microheterogeneity becomes unavoidable when the number of compartments is limited, or enzymes are excessively promiscuous.
| S-EPMC7360365 | biostudies-literature
Project description:Formation of covalent protein adducts by lipid electrophiles contributes to diseases and toxicities linked to oxidative stress, but analysis of the adducts presents a challenging analytical problem. We describe selective adduct capture using biotin affinity probes to enrich protein and peptide adducts for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). One approach employs biotinamidohexanoic acid hydrazide to covalently label residual carbonyl groups on adducts. The other employs alkynyl analogs of lipid electrophiles, which form adducts that can be postlabeled with azidobiotin tags by Cu(+)-catalyzed cycloaddition (Click chemistry). To enhance the selectivity of adduct capture, we use an azidobiotin reagent with a photocleavable linker, which allows recovery of adducted proteins and peptides under mild conditions. This approach allows both the identification of protein targets of lipid electrophiles and sequence mapping of the adducts.
| S-EPMC3811082 | biostudies-literature