Tutkus, M. | Search bioRxiv

bioRxiv2020

Aggregation-related quenching of LHCII in liposomes revealed by single-molecule spectroscopy

Incorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted LHCII. By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates on bound surface. Our results showed that fluorescence lifetime in bulk and of that for single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII-LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

biophysics↗

bioRxiv2020

Oriented Soft DNA Curtains for Single Molecule Imaging

Over the past twenty years, single-molecule methods have become extremely important for biophysical studies. These methods, in combination with new nanotechnological platforms, can significantly facilitate experimental design and enable faster data acquisition. A nanotechnological platform, which utilizes flow-stretch of immobilized DNA molecules, called DNA Curtains, is one of the best examples of such combinations. Here, we employed new strategies to fabricate a flow-stretch assay of stably immobilized and oriented DNA molecules using protein template-directed assembly. In our assay a protein template patterned on a glass coverslip served for directional assembly of biotinylated DNA molecules. In these arrays, DNA molecules were oriented to one another and maintained extended either by single- or both-ends immobilization to the protein templates. For oriented both-end DNA immobilization we employed heterologous DNA labeling and protein template coverage with the anti-digoxigenin antibody. In contrast to the single-end, both-ends immobilization does not require constant buffer flow for keeping DNAs in an extended configuration, allowing us to study protein-DNA interactions at more controllable reaction conditions. Additionally, we increased immobilization stability of the biotinylated DNA molecules using protein templates fabricated from traptavidin. Finally, we demonstrated that double-tethered Soft DNA Curtains can be used in nucleic acid-interacting protein (e.g. CRISPR-Cas9) binding assay that monitors binding location and position of individual fluorescently labeled proteins on DNA.

biophysics↗