Search Results (37)

Riboswitches regulate gene expression through intricate dynamic conformational transitions, with divalent cation Mg²⁺ and their ligands playing pivotal roles in this process. The dynamic structural mechanism by which the S-adenosyl-L-methionine (SAM) responsive SAM-VI riboswitch (riboSAM) regulates the downstream SAM synthase gene translation remains unclear. In this study, we employed position-selective labeling of RNA (PLOR) to incorporate Cy3-Cy5 into designated positions of riboSAM, applying single-molecule Förster resonance energy transfer (smFRET) method to track its conformational switches in response to Mg²⁺ and SAM. smFRET analysis revealed that in the absence of Mg²⁺ and ligand, riboSAM predominantly adopted a translation-activating apo conformation. Physiological concentrations of Mg²⁺ induced riboSAM to fold into dynamic transit-p and holo-p states, creating a transient and structurally pliable binding pocket for ligand binding. SAM binding locks the dynamic transit-p and holo-p states into their final stable transit and holo conformations through conformational selection, turning off downstream cis-gene expression and completing feedback regulation of cellular SAM concentration. The observed synergistic regulatory effect of Mg²⁺ ions and ligand on riboSAM’s conformational dynamics at single-molecule resolution provides new mechanistic insights into gene regulation by diverse riboswitch classes. Full article

Reconstituted high-density lipoprotein nanoparticles (NPs), which mimic the structure and function of endogenous human plasma HDL, hold promise as a robust drug delivery system. These nanoparticles, when loaded with appropriate agents, serve as powerful tools for targeted drug delivery. The fundamental challenge lies in controlling and estimating the actual drug load and the efficiency of drug release at the target. In this report, we present a novel approach based on enhanced Förster Resonance Energy Transfer (FRET) to assess particle load and monitor payload release. The NPs are labeled with donor molecules embedded in the lipid phase, while the spherical core volume is filled with acceptor molecules. Highly enhanced FRET efficiency to multiple acceptors in the NP core has been observed at distances significantly larger than the characteristic Förster distance (R₀). To confirm that the observed changes in donor and acceptor emissions are a result of FRET, we developed a theoretical model for nonradiative energy transfer from a single donor to multiple acceptors enclosed in a spherical core volume. The load-dependent shortening of the fluorescence lifetime of the donor correlated with the presence of a negative component in the intensity decay of the acceptor clearly demonstrates that FRET can occur at a large distance comparable to the nanoparticle size (over 100 Å). Comparison of theoretical simulations with the measured intensity decays of the donor and acceptor fluorophores constitute a new method for evaluating particle load. The observed FRET efficiency depends on the number of acceptors in the core, providing a simple way to estimate the nanoparticle load efficiency. Particle disintegration and load release result in a distinct change in donor and acceptor emissions. This approach constitutes a novel strategy for assessing NP core load, monitoring NP integrity, and evaluating payload release efficiency to target cells. Significants: In the last decade, nanoparticles have emerged as a promising strategy for targeted drug delivery, with applications ranging from cancer therapy to ocular neurodegenerative disease treatments. Despite their potential, a significant issue has been the real-time monitoring of these drug delivery vehicles within biological systems. Effective strategies for monitoring NP payload loading, NP integrity, and payload release are needed to assess the quality of new drug delivery systems. In our study, we have found that FRET-enabled NPs function as an improved method for monitoring these aspects currently missing from current drug delivery efforts. Full article

Receptor–ligand interactions at cell interfaces initiate signaling cascades essential for cellular communication and effector functions. Specifically, T cell receptor (TCR) interactions with pathogen-derived peptides presented by the major histocompatibility complex (pMHC) molecules on antigen-presenting cells are crucial for T cell activation. The binding duration, or dwell time, of TCR–pMHC interactions correlates with downstream signaling efficacy, with strong agonists exhibiting longer lifetimes compared to weak agonists. Traditional surface plasmon resonance (SPR) methods quantify 3D affinity but lack cellular context and fail to account for factors like membrane fluctuations. In the recent years, single-molecule Förster resonance energy transfer (smFRET) has been applied to measure 2D binding kinetics of TCR–pMHC interactions in a cellular context. Here, we introduce a rigorous mathematical model based on survival analysis to determine exponentially distributed receptor–ligand interaction lifetimes, verified through simulated data. Additionally, we developed a comprehensive analysis pipeline to extract interaction lifetimes from raw microscopy images, demonstrating the model’s accuracy and robustness across multiple TCR–pMHC pairs. Our new software suite automates data processing to enhance throughput and reduce bias. This methodology provides a refined tool for investigating T cell activation mechanisms, offering insights into immune response modulation. Full article

In this work, we apply single-molecule fluorescence microscopy and spectroscopy to probe plasmon-enhanced fluorescence and Förster resonance energy transfer in a nanoscale assemblies. The structure where the interplay between these two processes was present consists of photoactive proteins conjugated with silver nanowires and deposited on a monolayer graphene. By comparing the results of continuous-wave and time-resolved fluorescence microscopy acquired for this structure with those obtained for the reference samples, where proteins were coupled with either a graphene monolayer or silver nanowires, we find clear indications of the interplay between plasmonic enhancement and the energy transfer to graphene. Namely, fluorescence intensities calculated for the structure, where proteins were coupled to graphene only, are less than for the structure playing the central role in this study, containing both silver nanowires and graphene. Conversely, decay times extracted for the latter are shorter compared to a protein—silver nanowire conjugate, pointing towards emergence of the energy transfer. Overall, the results show that monitoring the optical properties of single emitters in a precisely designed hybrid nanostructure provides an elegant way to probe even complex combination of interactions at the nanoscale. Full article

A protein fold is defined as a structural arrangement of a secondary structure in a three-dimensional space. It would be interesting to know whether a particular fold can be assigned to certain features of the corresponding folding/unfolding transitions. To understand the underlying principles of the manifold folding transitions in more detail, single-molecule FRET is the method of choice. Taking the two-domain protein phosphoglycerate kinase (PGK) as an example, we investigated denaturant-induced unfolded states of PGK using the above method. For this purpose, different intramolecular distances within the two domains were measured. In addition to the known two-state transition, a transition with a compact folding intermediate was also identified in each of the two domains. Based on the structural homology of the domains (characterized by a Rossmann fold) and the striking similarity in the features of the measured distance changes during unfolding, clear evidence emerged that the underlying domain topology plays an important role in determining the observed structural changes. Full article

F_oF₁-ATP synthases in mitochondria, in chloroplasts, and in most bacteria are proton-driven membrane enzymes that supply the cells with ATP made from ADP and phosphate. Different control mechanisms exist to monitor and prevent the enzymes’ reverse chemical reaction of fast wasteful ATP hydrolysis, including mechanical or redox-based blockade of catalysis and ADP inhibition. In general, product inhibition is expected to slow down the mean catalytic turnover. Biochemical assays are ensemble measurements and cannot discriminate between a mechanism affecting all enzymes equally or individually. For example, all enzymes could work more slowly at a decreasing substrate/product ratio, or an increasing number of individual enzymes could be completely blocked. Here, we examined the effect of increasing amounts of ADP on ATP hydrolysis of single Escherichia coli F_oF₁-ATP synthases in liposomes. We observed the individual catalytic turnover of the enzymes one after another by monitoring the internal subunit rotation using single-molecule Förster resonance energy transfer (smFRET). Observation times of single FRET-labeled F_oF₁-ATP synthases in solution were extended up to several seconds using a confocal anti-Brownian electrokinetic trap (ABEL trap). By counting active versus inhibited enzymes, we revealed that ADP inhibition did not decrease the catalytic turnover of all F_oF₁-ATP synthases equally. Instead, increasing ADP in the ADP/ATP mixture reduced the number of remaining active enzymes that operated at similar catalytic rates for varying substrate/product ratios. Full article

Förster resonance energy transfer (FRET)-based biosensors are being fabricated for specific detection of biomolecules or changes in the microenvironment. FRET is a non-radiative transfer of energy from an excited donor fluorophore molecule to a nearby acceptor fluorophore molecule. In a FRET-based biosensor, the donor and acceptor molecules are typically fluorescent proteins or fluorescent nanomaterials such as quantum dots (QDs) or small molecules that are engineered to be in close proximity to each other. When the biomolecule of interest is present, it can cause a change in the distance between the donor and acceptor, leading to a change in the efficiency of FRET and a corresponding change in the fluorescence intensity of the acceptor. This change in fluorescence can be used to detect and quantify the biomolecule of interest. FRET-based biosensors have a wide range of applications, including in the fields of biochemistry, cell biology, and drug discovery. This review article provides a substantial approach on the FRET-based biosensor, principle, applications such as point-of-need diagnosis, wearable, single molecular FRET (smFRET), hard water, ions, pH, tissue-based sensors, immunosensors, and aptasensor. Recent advances such as artificial intelligence (AI) and Internet of Things (IoT) are used for this type of sensor and challenges. Full article

Translational G proteins, whose release from the ribosome is triggered by GTP hydrolysis, regulate protein synthesis. Concomitantly with binding and dissociation of protein factors, translation is accompanied by forward and reverse rotation between ribosomal subunits. Using single-molecule measurements, we explore the ways in which the binding of translational GTPases affects inter-subunit rotation of the ribosome. We demonstrate that the highly conserved translation factor LepA, whose function remains debated, shifts the equilibrium toward the non-rotated conformation of the ribosome. By contrast, the catalyst of ribosome translocation, elongation factor G (EF-G), favors the rotated conformation of the ribosome. Nevertheless, the presence of P-site peptidyl-tRNA and antibiotics, which stabilize the non-rotated conformation of the ribosome, only moderately reduces EF-G binding. These results support the model suggesting that EF-G interacts with both the non-rotated and rotated conformations of the ribosome during mRNA translocation. Our results provide new insights into the molecular mechanisms of LepA and EF-G action and underscore the role of ribosome structural dynamics in translation. Full article

Chromatins are structural components of chromosomes and consist of DNA and histone proteins. The structure, dynamics, and function of chromatins are important in regulating genetic processes. Several different experimental and theoretical tools have been employed to understand chromatins better. In this review, we will focus on the literatures engrossed in understanding of chromatins using single-molecule Förster resonance energy transfer (smFRET). smFRET is a single-molecule fluorescence microscopic technique that can furnish information regarding the distance between two points in space. This has been utilized to efficiently unveil the structural details of chromatins. Full article

Single-stranded DNA-binding proteins (SSBs) play vital roles in DNA metabolism. Proteins of the SSB family exclusively and transiently bind to ssDNA, preventing the DNA double helix from re-annealing and maintaining genome integrity. In the meantime, they interact and coordinate with various proteins vital for DNA replication, recombination, and repair. Although SSB is essential for DNA metabolism, proteins of the SSB family have been long described as accessory players, primarily due to their unclear dynamics and mechanistic interaction with DNA and its partners. Recently-developed single-molecule tools, together with biochemical ensemble techniques and structural methods, have enhanced our understanding of the different coordination roles that SSB plays during DNA metabolism. In this review, we discuss how single-molecule assays, such as optical tweezers, magnetic tweezers, Förster resonance energy transfer, and their combinations, have advanced our understanding of the binding dynamics of SSBs to ssDNA and their interaction with other proteins partners. We highlight the central coordination role that the SSB protein plays by directly modulating other proteins’ activities, rather than as an accessory player. Many possible modes of SSB interaction with protein partners are discussed, which together provide a bigger picture of the interaction network shaped by SSB. Full article

Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg²⁺ but accelerates the transition rate at 10 mM Mg²⁺. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms. Full article

Single-molecule Förster resonance energy transfer (smFRET) enables the deconvolution of various conformational substates of biomolecules. Over the past two decades, it has been widely used to understand the conformational dynamics of enzymes. Commonly, enzymes undergo reversible transitions between active and inactive states in solution. Using smFRET, the details of these transitions and the effect of ligands on these dynamics have been determined. In this mini-review, we discuss the various works focused on the investigation of enzyme conformational dynamics using smFRET. Full article

For single-molecule studies in solution, very small concentrations of dye-labelled molecules are employed in order to achieve single-molecule sensitivity. In typical studies with confocal microscopes, often concentrations in the pico-molar regime are required. For various applications that make use of single-molecule Förster resonance energy transfer (smFRET) or two-color coincidence detection (TCCD), the molecule concentration must be set explicitly to targeted values and furthermore needs to be stable over a period of several hours. As a consequence, specific demands must be imposed on the surface passivation of the cover slides during the measurements. The aim of having only one molecule in the detection volume at the time is not only affected by the absolute molecule concentration, but also by the rate of diffusion. Therefore, we discuss approaches to control and to measure absolute molecule concentrations. Furthermore, we introduce an approach to calculate the probability of chance coincidence events and demonstrate that measurements with challenging smFRET samples require a strict limit of maximal sample concentrations in order to produce meaningful results. Full article

Single molecule fluorescence and nuclear magnetic resonance spectroscopy (NMR) are two very powerful techniques for the analysis of intrinsically disordered proteins (IDPs). Both techniques have individually made major contributions to deciphering the complex properties of IDPs and their interactions, and it has become evident that they can provide very complementary views on the distance-dynamics relationships of IDP systems. We now review the first approaches using both NMR and single molecule fluorescence to decipher the molecular properties of IDPs and their interactions. We shed light on how these two techniques were employed synergistically for multidomain proteins harboring intrinsically disordered linkers, for veritable IDPs, but also for liquid–liquid phase separated systems. Additionally, we provide insights into the first approaches to use single molecule Förster resonance energy transfer (FRET) and NMR for the description of multiconformational models of IDPs. Full article

Membrane proteins have evolved to work optimally within the complex environment of the biological membrane. Consequently, interactions with surrounding lipids are part of their molecular mechanism. Yet, the identification of lipid–protein interactions and the assessment of their molecular role is an experimental challenge. Recently, biophysical approaches have emerged that are compatible with the study of membrane proteins in an environment closer to the biological membrane. These novel approaches revealed specific mechanisms of regulation of membrane protein function. Lipids have been shown to play a role in oligomerization, conformational transitions or allosteric coupling. In this review, we summarize the recent biophysical approaches, or combination thereof, that allow to decipher the role of lipid–protein interactions in the mechanism of membrane proteins. Full article