Biology subjects

Tych, K.

Publications and source records attributed to Tych, K..

5 recordsLinked to original sources

bioRxiv2024

One-Pot Dual Protein Labelling for Simultaneous Mechanical and Fluorescent Readouts in Optical Tweezers

Optical tweezers are widely used in the study of biological macromolecules but are limited by their one-directional probing capability, potentially missing critical conformational changes. Combining fluorescence microscopy with optical tweezers, employing Forster resonance energy transfer (FRET) pairs, addresses this issue. Moreover, attaching one FRET probe to a tethered protein and the other to a protein in solution allows precise localisation of interaction sites, while probing mechanical properties. When integrating fluorescence microscopy with optical tweezers, orthogonal protein conjugation methods are needed to enable simultaneous, site-specific attachment of fluorophores and DNA handles, commonly used to apply force to molecules of interest. In this study, we utilized commercially available reagents for dual site-specific labelling of the homodimeric heat shock protein 90 (Hsp90) using thiol-maleimide and inverse electron demand Diels-Alder cycloaddition (IEDDAC) bioorthogonal reactions. In a one-pot approach, Hsp90 modified with a cysteine mutation and the non-canonical amino acid cyclopropene-L-lysine (CpK) was labelled with the FRET pair maleimide-Atto550 and maleimide-Atto647N, alongside single- stranded methyltetrazine-modified DNA oligonucleotide. Optical tweezers experiments with this labelled Hsp90 construct revealed structural transitions consistent with previous studies, validating the approach. Fluorescence measurements confirmed the proximity of FRET pairs in the N-terminally closed state of Hsp90 in this experimental setup. This integrative method provides a powerful tool for probing protein conformational dynamics and protein interactions beyond the limitations of traditional optical tweezers. StatementThe developed method combines fluorescence microscopy with optical tweezers, enhancing single-molecule protein studies by overcoming the limitations of one- directional mechanical probing. Utilizing two orthogonal protein conjugation methods for one-pot dual labelling, the heat shock protein 90 was labelled with a FRET pair and single-stranded DNA oligonucleotides. Validated by comparison with published conformational changes, mechanical unfolding signatures, and FRET pair distances, this approach provides a powerful tool to explore single-molecule conformational dynamics and protein interactions.

biophysics↗

bioRxiv2024

Hybrid lipid-block copolymer membranes enable stable reconstitution of a wide range of nanopores and robust sampling of serum

Biological nanopores are powerful tools for detecting biomolecules at the single-molecule level, making them appealing as sensors for biological samples. However, the lipid membranes in which nanopores reside can be unstable in the presence of biological fluids. Here, membranes formed with the amphiphilic polymers PMOXA-PDMS-PMOXA and PBD-PEO are tested as potential alternatives for nanopore sensing. We demonstrate that polymer membranes can possess increased stability towards applied potentials and high concentrations of human serum, but that the stable insertion of a wide range of biological nanopores is most often compromised. Alternatively, hybrid polymer-lipid membranes comprising a 1:1 w/w mixture of PBD11PEO8 and DPhPC showed high electrical and biochemical stability while creating a suitable environment for all tested nanopores. Analytes such as proteins, DNA and sugars were efficiently sampled, indicating that in hybrid membranes nanopores showed native-like properties. Molecular dynamics simulations revealed that lipids form [~]12 nm domains interspersed by a polymer matrix. Nanopores partitioned into these lipid nanodomains and sequestered lipids possibly offering the same binding strength as in a native bilayer. This work shows that single-molecule analysis with nanopores in [PBD11PEO8 + DPhPC] membranes is feasible and present stable recordings in the presence of human serum. These results pave the way towards novel nanopore-based biosensors.

biophysics↗

bioRxiv2024

Investigating the mechanisms of enzyme diffusion using mass photometry

The existence of the phenomenon of enhanced enzyme diffusion (EED) has been a topic of debate in recent literature. One proposed mechanism to explain the origin of EED is oligomeric enzyme dissociation. We use mass photometry (MP), a label-free single-molecule technique, to investigate the dependence of the oligomeric states of several enzymes on their ligands. The studied enzymes of interest are catalase, aldolase, alkaline phosphatase and vanillyl-alcohol oxidase (VAO). We compared the ratios of oligomeric states in the presence and absence of substrate as well as different substrate and inhibitor concentrations. Catalase and aldolase were found to dissociate into smaller oligomers in the presence of their substrates, independently of inhibition, while for alkaline phosphatase and VAO, different behaviors were observed. Thus, we have identified a possible mechanism which explains the previously observed diffusion enhancement in vitro. This enhancement may occur due to the dissociation of oligomers through ligand binding.

biophysics↗

bioRxiv2023

Probing the stability and interdomain interactions in the ABC transporter OpuA, using single-molecule optical tweezers

Transmembrane transporter proteins are essential for the maintenance of cellular homeostasis and, as such, are key drug targets. Many transmembrane transporter proteins are known to undergo large structural rearrangements during their functional cycles. Despite the large amount of detailed structural and functional data available for these systems, our understanding of their dynamics and therefore how they function is generally limited. We introduce an innovative approach which enables us to directly measure the dynamics and stabilities of inter-domain interactions of transmembrane proteins using optical tweezers. Focusing on the osmoregulatory ABC transporter OpuA from Lactococcus lactis, we examine the mechanical properties and potential interactions of its substrate-binding domains. Our measurements are performed in lipid nanodiscs, providing a native-mimicking environment for the transmembrane protein. The technique provides high spatial and temporal resolution and allows us to study the functionally-relevant motions and inter-domain interactions of individual transmembrane transporter proteins in real-time in a lipid bilayer.

biophysics↗

bioRxiv2023

Biophysical analysis reveals autophosphorylation as an important negative regulator of LRRK2 dimerization

Leucine-rich repeat kinase 2 (LRRK2) is a large, multi-domain protein which is associated with Parkinsons disease. Although high-resolution structures of LRRK2 are available, little is known about the complex dynamics behind the inter-domain regulation of LRRK2 and its perturbation by pathogenic variants. Previous studies have demonstrated that LRRK2 goes through an oligomerization cycle at the membrane, however it remains unclear in which form it exerts its kinase activity. Moreover, the LRRK2 monomer-dimer equilibrium and associated functional implications at a molecular level also need further investigation. In the present work, we used a multi-faceted approach to better understand LRRK2 oligomerization and suggest a functional model of how LRRK2 interacts with its substrates. To this end, we combined nano differential scanning calorimetry and mass photometry with molecular modelling. The thermal analysis resulted in a multistep denaturation profile, elucidating novel insights into the composite structural organization of the multi-domain protein LRRK2. Furthermore, LRRK2 shows a remarkable thermal stability, confirming its oligomeric nature. By using mass photometry, we could observe a monomer-dimer equilibrium which is altered by R1441G, a pathogenic variant within the Roc-COR interface. Most importantly, we could demonstrate that autophosphorylation induces LRRK2 monomerization, indicating a novel intramolecular feedback mechanism. Finally, we investigated the interaction of LRRK2 with its substrate, RAB10 by integrative computational modelling. The resulting models suggest that the monomeric form of LRRK2 is the favored protein conformation for the interaction with its substrate, leading to an increasing interest in the monomer-dimer equilibrium as a possible intervention point for the pathology.

biochemistry↗