Biology subjects

Olesen, C. H.

Publications and source records attributed to Olesen, C. H..

4 recordsLinked to original sources

bioRxiv2026

HaloTag Ligand and HaloTag Protein engineering for a binary fluorescent turn-on probe

Protein labelling by covalent attachment of a specific substrate to a self-labelling protein tag has become a regular in the life sciences. Herein, we report the design of a two-component labelling system, comprised of a non-fluorescent difluorinated xanthene, called F2X, and a HaloTag mutant engineered for targeted reactivity towards F2X. Upon primary covalent locking of the ligand at the canonical aspartate residue, two proximal lysine residues located at the protein surface can undergo nucleophilic aromatic substitution with the F2X core, building a fluorescent rhodamine via triple-covalent fusion. We used a generalizable in silico pipeline for heuristic conformational sampling of covalent protein-ligand complexes to find suitable mutation sites, culminating in the curation of 7 double-lysine HaloTag mutants for targeted in vitro testing. Reaction with the best-performing mutant, HTPL161K_Q165K, is characterized by full protein mass spectrometry, fluorescence polarization fluorescence lifetime, and fluorescence anisotropy and rationalized by computational modelling. We showcase the system in single molecule microscopy, where obviation of post-labelling purification is a prime advantage when targeting recombinant proteins that may not be expressed in larger quantities, and employ F2X in living cells with reduced photobleaching. Lastly, a cell-impermeable version was obtained by means of sulfonation, exclusively targeting extracellularly exposed HTPKK fused to the neuromodulatory G protein-coupled receptor metabotropic glutamate receptor 2.

synthetic biology↗

bioRxiv2026

A bio-orthogonal and covalent 5 kDa small protein tag

Precise and minimally perturbative protein labelling remains a key challenge for studying biomolecular function in living systems. Here, a minimal way to specifically label proteins based on SNAP-tag and intein-mediated protein splicing reaction is introduced. Termed CLUSTER (for Chemical Label-Unfold-Splice Technology Enables Recombination), this chimeric platform supports efficient labelling across diverse targets in living cells by retaining a fluorescent, 5 kDa sized peptide on a fusion protein of interest after splicing. A bacterial screening workflow was developed to optimize the reaction efficiency and construct design. Quantitative characterization using fluorescence polarization provides mechanistic insight into labelling efficiency and dynamics, while molecular dynamics simulations elucidate its stability, grasping the intricate nature of protein behaviour upon covalent labelling. This bio-orthogonal labelling technology allows for a versatile and minimally invasive approach for protein labelling, providing a powerful tool to probe protein behavior in native cellular systems.

synthetic biology↗

bioRxiv2024

A one-step protocol to generate impermeable fluorescent HaloTag substrates for in situ live cell application and super-resolution imaging

Communication between cells is largely orchestrated by proteins on the cell surface, which allow information transfer across the cell membrane. Super-resolution and single-molecule visualization of these proteins can be achieved by genetically grafting HTP (HaloTag Protein) into the protein of interest followed by brief incubation of cells with a dye-HTL (dye-linked HaloTag Ligand). This approach allows for use of cutting-edge fluorophores optimized for specific optical techniques or a cell-impermeable dye-HTL to selectively label surface proteins without labeling intracellular copies. However, these two goals often conflict, as many high-performing dyes exhibit membrane permeability. Traditional methods to eliminate cell permeability face synthetic bottlenecks and risk altering photophysical properties. Here we report that dye-HTL reagents can be made cell-impermeable by inserting a charged sulfonate directly into the HTL, leaving the dye moiety unperturbed. This simple, one-step method requires no purification and is compatible with both the original HTL and second-generation HTL.2, the latter offering accelerated labeling. We validate such compounds, termed dye-SHTL ( dye shuttle) conjugates, in live cells via widefield microscopy, demonstrating exclusive membrane staining of extracellular HTP fusion proteins. In transduced primary hippocampal neurons, we label mGluR2, a neuromodulatory G protein-coupled receptor (GPCR), with dyes optimized for stimulated emission by depletion (STED) super-resolution microscopy, allowing unprecedented accuracy in distinguishing surface and receptors from those in internal compartments of the presynaptic terminal, important in neural communication. This approach offers broad utility for surface-specific protein labelling.

pharmacology and toxicology↗

bioRxiv2023

Structure of the hepatitis C virus E1/E2 envelope proteins in a homodimeric complex

Worldwide, 58 million individuals suffer from chronic hepatitis C virus (HCV) infection, a primary driver of liver cancer. The HCV envelope proteins, E1 and E2, form a heterodimer, which is the target for neutralizing antibodies. Despite high-resolution structural models of partial heterodimer elements, the structural landscape of higher-order E1/E2 oligomers remains unexplored. We determined a ~3.5 [A] cryo-electron microscopy structure of membrane-extracted HCV E1/E2 in a homodimeric arrangement. This structure includes detailed information on the homodimer interface, the E2-binding pocket for hypervariable region 1, antigenic site 412 conformation, and the organization of the E1/E2 transmembrane regions, including one internal to E1. This higher-order E1/E2 assembly could play a pivotal role in the design of novel vaccine antigens better mimicking E1/E2 complexes on the HCV particle.

molecular biology↗