Fluorescence Microscopy of Single Liposomes with Incorporated Pigment-Proteins

Reconstitution of transmembrane proteins into liposomes is a widely used method to study their behavior under conditions closely resembling the natural ones. However, this approach does not allow precise control of the liposome size, reconstitution efficiency, and the actual protein-to-lipid ratio in the formed proteoliposomes, which might be critical for some applications and/or interpretation of data acquired during the spectroscopic measurements. Here, we present a novel strategy employing methods of proteoliposome preparation, fluorescent labeling, purification, and surface immobilization that allow us to quantify these properties using fluorescence microscopy at the singleliposome level and for the first time apply it to study photosynthetic pigment protein complexes LHCII. We show that LHCII proteoliposome samples, even after purification with a density gradient, always contain a fraction of nonreconstituted protein and are extremely heterogeneous in both protein density and liposome sizes. This strategy enables quantitative analysis of the reconstitution efficiency of different protocols and precise fluorescence spectroscopic study of various transmembrane proteins in a controlled nativelike environment

Pavienių molekulių fluorescencinė mikroskopija baltymų dinamikos tyrimams

Conformational dynamics of proteins are essential for their functioning objectives, which are possible due to the inherent flexibility of protein structure. Every protein, regardless of whether it is located in lipid membranes or found in the cytosol undergoes conformational dynamics. The mentioned classes of proteins are different regarding their native environment and involved in different aspects of cellular life. In this work we chose to investigate the following representatives from two different classes – transmembrane pigment-protein complexes from photosystem II of plants and water-soluble proteins interacting with nucleic acids – DNA Restriction endonucleases. Since conformational dynamics of protein are typically hidden in an ensemble type of measurements we revealed them with the aid of the single-molecule methods. Our results on single-molecule conformational dynamics studies of protein-pigment complexes and DNA-interacting proteins provided essential insights into each field of research. Also, both of our developed methods were successfully applied and will be useful for future studies of various DNA-interacting proteins and other pigment-protein complexes or their assemblies

DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

Protein-DNA interactions are the core of the cell’s molecular machinery. For a long time, conventional biochemical methods served as a powerful investigatory basis of protein-DNA interactions and target search mechanisms. Currently single-molecule (SM) techniques have emerged as a complementary tool for studying these interactions and have revealed plenty of previously obscured mechanistic details. In comparison to the traditional ones, SM methods allow direct monitoring of individual biomolecules. Therefore, SM methods reveal reactions that are otherwise hidden by the ensemble averaging observed in conventional bulk-type methods. SM biophysical techniques employing various nanobiotechnology methods for immobilization of studied molecules grant the possibility to monitor individual reaction trajectories of biomolecules. Next-generation in vitro SM biophysics approaches enabling high-throughput studies are characterized by much greater complexity than the ones developed previously. Currently, several high-throughput DNA flow-stretch assays have been published and have shown many benefits for mechanistic target search studies of various DNA-binding proteins, such as CRISPR-Cas, Argonaute, various ATP-fueled helicases and translocases, and others. This review focuses on SM techniques employing surface-immobilized and relatively long DNA molecules for studying protein-DNA interaction mechanisms

DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

Protein-DNA interactions are the core of the cell’s molecular machinery. For a long time, conventional biochemical methods served as a powerful investigatory basis of protein-DNA interactions and target search mechanisms. Currently single-molecule (SM) techniques have emerged as a complementary tool for studying these interactions and have revealed plenty of previously obscured mechanistic details. In comparison to the traditional ones, SM methods allow direct monitoring of individual biomolecules. Therefore, SM methods reveal reactions that are otherwise hidden by the ensemble averaging observed in conventional bulk-type methods. SM biophysical techniques employing various nanobiotechnology methods for immobilization of studied molecules grant the possibility to monitor individual reaction trajectories of biomolecules. Next-generation in vitro SM biophysics approaches enabling high-throughput studies are characterized by much greater complexity than the ones developed previously. Currently, several high-throughput DNA flow-stretch assays have been published and have shown many benefits for mechanistic target search studies of various DNA-binding proteins, such as CRISPR-Cas, Argonaute, various ATP-fueled helicases and translocases, and others. This review focuses on SM techniques employing surface-immobilized and relatively long DNA molecules for studying protein-DNA interaction mechanisms

TGF beta-induced changes in membrane curvature influence Ras oncoprotein membrane localization

In the course of cancer progression tumor cells undergo morphological changes that lead to increased motility and invasiveness thus promoting formation of metastases. This process called epithelial to mesenchymal transition (EMT) is triggered by transforming growth factor (TGFβ) but for gaining the full invasive potential an interplay between signaling of TGFβ and Ras GTPases is required. Ras proteins possess a lipidated domain that mediates Ras association with the plasma membrane, which is essential for Ras biological functions. Type and number of the lipid anchors are the main difference among three Ras variants—H-ras, N-ras and K-ras. The lipid anchors determine membrane partitioning of lipidated proteins into membrane areas of specific physico-chemical properties and curvature. In this study, we investigated the effect of TGFβ treatment on the subcellular localization of H-ras and K-ras. We show that TGFβ increases positive plasma membrane curvature, which is subsequently sensed by H-ras, leading to its elevated plasma membrane localization and activation. This observation suggests the existence of a novel positive feedback loop whereby the increased level of plasma membrane curvature during TGFβ induced EMT attracts more Ras molecules to the plasma membrane resulting in increased Ras activity which in turn promotes further EMT and thus ultimately enables the acquisition of full invasive potential

The miEye: bench-top super-resolution microscope with cost-effective equipment

Commercial super-resolution (SR) imaging systems require a high budget, while current more affordable open source microscopy systems lack modularity and sometimes are too complex or lack reliability. We present miEye - a cost-effective microscope designed for high-resolution wide-field fluorescence imaging. The build is constructed using a CNC milled aluminum microscope body and commercially available optomechanics, with open-source Python-based microscope control, data visualization, and analysis software integration. The data acquisition software works robustly with commonly used industrial-grade complementary metal oxide semiconductor (iCMOS) cameras, performs IR beam back-reflection-based automatic focus stabilization, and allows for laser control via an Arduino-based laser relay. The open-source nature of the design is aimed to facilitate adaptation by the community. The build can be constructed for a cost of roughly 50 k €. It contains SM-fiber and MM-fiber excitation paths that are easy to interchange and an adaptable emission path. Also, it ensures <5 nm/min stability of the sample on all axes, and allows achieving <30 nm lateral resolution for dSTORM and DNA-PAINT single-molecule localization microscopy (SMLM) experiments. Thus it serves as a cost-effective and adaptable addition to the open source microscopy community and potentially will allow high-quality SR imaging even for limited-budget research groups

Aggregation-related quenching of LHCII fluorescence 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 major light-harvesting complexes of plants (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 bound to the surface. Our results showed that fluorescence lifetime obtained in bulk and in 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. © 2021 Elsevier B.V

Disarming of type I-F CRISPR-Cas surveillance complex by anti-CRISPR proteins AcrIF6 and AcrIF9

CRISPR-Cas systems are prokaryotic adaptive immune systems that protect against phages and other invading nucleic acids. The evolutionary arms race between prokaryotes and phages gave rise to phage anti-CRISPR (Acr) proteins that act as a counter defence against CRISPR-Cas systems by inhibiting the effector complex. Here, we used a combination of bulk biochemical experiments, X-ray crystallography and single-molecule techniques to explore the inhibitory activity of AcrIF6 and AcrIF9 proteins against the type I-F CRISPR-Cas system from Aggregatibacter actinomycetemcomitans (Aa). We showed that AcrIF6 and AcrIF9 proteins hinder Aa-Cascade complex binding to target DNA. We solved a crystal structure of Aa1-AcrIF9 protein, which differ from other known AcrIF9 proteins by an additional structurally important loop presumably involved in the interaction with Cascade. We revealed that AcrIF9 association with Aa-Cascade promotes its binding to off-target DNA sites, which facilitates inhibition of CRISPR-Cas protection

Fixed DNA Molecule Arrays for High-Throughput Single DNA-Protein Interaction Studies

The DNA Curtains assay is a recently developed experimental platform for protein-DNA interaction studies at the single-molecule level that is based on anchoring and alignment of DNA fragments. The DNA Curtains so far have been made by using chromium barriers and fluid lipid bilayer membranes, which makes such a specialized assay technically challenging and relatively unstable. Herein, we report on an alternative strategy for DNA arraying for analysis of individual DNA-protein interactions. It relies on stable DNA tethering onto nano-patterned protein templates via high affinity molecular recognition. We describe fabrication of streptavidin templates (line features as narrow as 200 nm) onto modified glass coverslips by combining surface chemistry, atomic force microscopy (AFM), and soft lithography techniques with affinity-driven assembly. We have employed such chips for arraying single- and double-tethered DNA strands, and we characterized the obtained molecular architecture: we evaluated the structural characteristics and specific versus nonspecific binding of fluorescence-labeled DNA using AFM and total internal reflection fluorescence microscopy. We demonstrate the feasibility of our DNA molecule arrays for short single-tethered as well as for lambda single- and double-tethered DNA. The latter type of arrays proved very suitable for localization of single DNA-protein interactions employing restriction endonucleases. The presented molecular architecture and facile method of fabrication of our nanoscale platform does not require clean room equipment, and it offers advanced functional studies of DNA machineries and the development of future nanodevices