Traceless cysteine-linchpin enables precision engineering of lysine in native proteins.

The maintenance of machinery requires its operational understanding and a toolbox for repair. The methods for the precision engineering of native proteins meet a similar requirement in biosystems. Its success hinges on the principles regulating chemical reactions with a protein. Here, we report a technology that delivers high-level control over reactivity, chemoselectivity, site-selectivity, modularity, dual-probe installation, and protein-selectivity. It utilizes cysteine-based chemoselective Linchpin-Directed site-selective Modification of lysine residue in a protein (LDMC-K). The efficiency of the end-user-friendly protocol is evident in quantitative conversions within an hour. A chemically orthogonal C-S bond-formation and bond-dissociation are essential among multiple regulatory attributes. The method offers protein selectivity by targeting a single lysine residue of a single protein in a complex biomolecular mixture. The protocol renders analytically pure single-site probe-engineered protein bioconjugate. Also, it provides access to homogeneous antibody conjugates (AFC and ADC). The LDMC-K-ADC exhibits highly selective anti-proliferative activity towards breast cancer cells.

Electrochemical biosensors based on peptide-kinase interactions at the kinase docking site.

Kinases are important cancer biomarkers and are conventionally detected based on their catalytic activity. Kinases regulate cellular activities by phosphorylation of motif-specific multiple substrate proteins, resulting in a lack of selectivity of activity-based kinase biosensors. We present an alternative approach of sensing kinases based on the interactions of their allosteric docking sites with a specific partner protein. The new approach was demonstrated for the ERK2 kinase and its substrate ELK-1. A peptide derived from ELK-1 was bound to a gold electrode and ERK2 sensing was performed by electrochemical impedance spectroscopy. We performed a detailed analysis of the interaction between the ELK-1 peptide and the kinase on gold surfaces. Atomic force microscopy, variable angle spectroscopic ellipsometry, X-ray Photoelectron Spectroscopy, and polarization modulation IR reflection-absorption spectroscopy analysis of the gold surface revealed the adsorbed layer of the ERK2 on the peptide monolayer. The sensors showed a high level of target selectivity for ERK2 compared to the p38γ kinase and BSA. ERK2 was detected in its cellular concentration range, 0.5-2.0 µM, and the limit of detection was calculated to be 0.35 µM. Using the flexibility of peptide design, our method is generic for developing sensitive and substrate-specific biosensors and other disease-related enzymes based on their interactions.

Single-site labeling of histidine in proteins, on-demand reversibility, and traceless metal-free protein purification.

A precision methodology distinguishes one His from all the nucleophilic residues and its multiple copies. An easy-to-operate C-N bond formation labels diverse proteins without adversely affecting their structure and function. The late-stage transformation allows installation of distinct probes. The chemically triggered reversibility enables traceless metal-free purification of proteins with a His-tag.

A phthalimidation protocol that follows protein defined parameters.

This work outlines the first phthalimidation protocol suitable for protein labeling and performed in aqueous media at room temperature and neutral pH with no catalyst or co-reagent required. The methodology is suitable for a range of amines and its efficiency was determined with chemoselective and site-selective protein labeling.