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Single-molecule techniques provide insights into the heterogeneity and dynamics of ensembles and enable the extraction of mechanistic information that is complementary to high-resolution structural techniques. Here, we describe the application of single-molecule Förster resonance energy transfer to study the dynamics of integral membrane protein complexes on timescales spanning sub-milliseconds to minutes (10

Conventional biophysical and chemical biology approaches for delineating relationships between the structure and biological function of nucleic acids (NAs) abstract NAs from their native biological context. However, cumulative experimental observations have revealed that the structure, dynamics and interactions of NAs might be strongly influenced by a broad spectrum of specific and nonspecific physical-chemical environmental factors. This consideration has recently sparked interest in the development of novel tools for structural characterization of NAs in the native cellular context. Here, we review the individual methods currently being employed for structural characterization of NA structure in a native cellular environment with a focus on recent advances and developments in the emerging fields of in-cell NMR and electron paramagnetic resonance spectroscopy and in-cell single-molecule FRET of NAs.

C‐rich DNA has the capacity to form a tetra‐stranded structure known as an i‐motif. The i‐motifs within genomic DNA have been proposed to contribute to the regulation of DNA transcription. However, direct experimental evidence for the existence of these structures in vivo has been missing. Whether i‐motif structures form in complex environment of living cells is not currently known. Herein, using state‐of‐the‐art in‐cell NMR spectroscopy, we evaluate the stabilities of i‐motif structures in the complex cellular environment. We show that i‐motifs formed from naturally occurring C‐rich sequences in the human genome are stable and persist in the nuclei of living human cells. Our data show that i‐motif stabilities in vivo are generally distinct from those in vitro. Our results are the first to interlink the stability of DNA i‐motifs in vitro with their stability in vivo and provide essential information for the design and development of i‐motif‐based DNA biosensors for intracellular applications.

The Sec translocon is a transmembrane assembly highly conserved among all forms of life as the principal route for transport of polypeptides across or into lipid bilayers. In bacteria translocation involves allosteric communication between the membrane pore SecYEG and the associated SecA ATPase. Using singlemolecule fluorescence we reveal that slow conformational changes associated with the ATPase SecA modulate fast opening and closure of the SecY lateral gate. Such a mismatch of timescales is not compatible with direct coupling between SecA and SecYEG. A dynamic allosteric model is proposed in which the SecA ATPase cycle ‘steers’ the energy landscape for SecY pore opening. We map the experimental traces onto reduced reaction coordinates derived from molecular dynamics trajectories, providing a model for the energy landscape and a structural interpretation of the associated dynamics. Dynamic allostery may be common among motor ATPases that drive conformational changes in molecular machines.Graphical TOC Entry

Studies on DNA-ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring DNA-ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of NMR data from cells electroporated with preformed DNA-ligand complexes. The impact of the intracellular environment on the integrity of the complexes is assessed based on in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. This technique was tested on complexes of two model DNA fragments and four ligands, namely, a representative DNA minor-groove binder (netropsin) and ligands binding DNA base-pairing defects (naphthalenophanes). In the latter case, we demonstrate that two of the three in vitro-validated ligands retain their ability to form stable interactions with their model target DNA in cellulo, whereas the third one loses this ability due to off-target interactions with genomic DNA and cellular metabolites. Collectively, our data suggest that direct evaluation of the behavior of drug-like molecules in the intracellular environment provides important insights into the development of DNA-binding ligands with desirable biological activity and minimal side effects resulting from off-target binding.

High-resolution studies of DNA–ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this issue, we developed an in-cell NMR-based approach for monitoring DNA–ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of high-resolution NMR data of cells electroporated with pre-formed DNA-ligand complex. The impact of the intracellular environment on the integrity of the complex is assessed on the basis of in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. By using this technique, we studied complexes of model DNA fragments and four ligands, representative of DNA minor-groove binders (netropsin) or ligands binding to DNA pairing defects (naphthalenophanes). We demonstrate that some of the in vitro validated ligands retain their ability to form stable on-target DNA interactions in situ, while other lose this ability due to off-target interactions with genomic DNA as well as cellular metabolic components. Collectively, our data suggest that direct evaluation of behavior of drug-like molecules in the intracellular environment provides important insights for the design and development of DNA-binding ligands with the desired biological action and minimal side effects resulting from off-target binding.

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. This is mediated, for the majority of proteins, by the highly conserved Sec machinery. The bacterial translocon – SecYMKEG – resides in the plasma membrane, where translocation is driven through rounds of ATP hydrolysis by the cytoplasmic SecA ATPase, and the proton motive force (PMF). We have used single molecule Förster resonance energy transfer (FRET) alongside a combination of confocal and total internal reflection microscopy to gain access to SecY pore dynamics and translocation kinetics on timescales spanning milliseconds to minutes. This allows us to dissect and characterise the translocation process in unprecedented detail. We show that SecA, signal sequence, pre-protein and ATP hydrolysis each have important and specific roles in unlocking and opening the Sec channel, priming it for transport. After channel opening, translocation proceeds in two phases: an initiation phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~ 40 amino acids per second for the model pre-protein substrate proOmpA. The initiation and translocation phases are both coupled to ATP hydrolysis while termination is ATP-independent. Distributions of translocation rates reflect the stochastic nature of the translocation process and are consistent with the recently proposed Brownian ratchet model [Allenet al.doi: 10.7554/eLife.15598]. The results allow us unparalleled access to the kinetics of the complex reaction and provide a framework for understanding the molecular mechanism of protein secretion.

Chromatin-remodeling ATPases modulate histones-DNA interactions within nucleosomes and regulate transcription. At the heart of remodeling, ATPase is a helicase-like motor flanked by a variety of conserved targeting domains. CHD4 is the core subunit of the nucleosome remodeling and deacetylase complex NuRD and harbors tandem plant homeo finger (tPHD) and chromo (tCHD) domains. We describe a multifaceted approach to link the domain structure with function, using quantitative assays for DNA and histone binding, ATPase activity, shape reconstruction from solution scattering data, and single molecule translocation assays. These approaches are complementary to high-resolution structure determination.

Imaging of small objects such as single molecules, DNA clusters and single bacterial cells is problematic not only due to the lateral resolution that is obtainable in currently existing microscopy but also, and as much fundamentally limiting, due to the lack of sufficient axial depth of focus to have the full object focused simultaneously. Extension in depth of focus is helpful also for single molecule steady state FRET measurements. In this technique it is crucial to obtain data from many well focused molecules, which are often located in different axial depths. In this paper we present the implementation of an all-optical and a real time technique of extension in the depth of focus that may be incorporated in any high NA microscope system and to be used for the above mentioned applications. We demonstrate experimentally how after the integration of special optical element in high NA 100× objective lens of a single molecule imaging microscope system, the depth of focus is significantly improved while maintaining the same lateral resolution in imaging applications of incorporated groups of molecules, DNA constructs and clusters inside bacterial cells.

Chromatin-remodeling ATPases modulate histones-DNA interactions within nucleosomes and regulate transcription. At the heart of remodeling, ATPase is a helicase-like motor flanked by a variety of conserved targeting domains. CHD4 is the core subunit of the nucleosome remodeling and deacetylase complex NuRD and harbors tandem plant homeo finger (tPHD) and chromo (tCHD) domains. We describe a multifaceted approach to link the domain structure with function, using quantitative assays for DNA and histone binding, ATPase activity, shape reconstruction from solution scattering data, and single molecule translocation assays. These approaches are complementary to high-resolution structure determination.

Molecular dynamics (MD) simulation and ab initio quantum mechanical calculations were employed to investigate the structure and dynamics of binding of QSY 21 nonfluorescent quencher to B-DNA decamer. Two binding motifs were observed: 1. Central xanthone ring of QSY 21 dye is oriented paralelly with the nearest DNA base pair and the complex is stabilized by stacking interaction between these two components. This binding motif has two substates. 2. The QSY 21 dye stacking interaction with the DNA bases is mediated only by one of the 2,3-dihydro-1-indolyl side rings of the dye. The ab initio calculations show that none of these binding motifs is energetically preferred. The less favorable stacking interaction between the dye and DNA bases in the second binding motif is probably compensated by advantageous interaction of dye and linker with DNA backbone.

The k-turn is a commonly occurring structural motif that introduces a tight kink into duplex RNA. In free solution, it can exist in an extended form, or by folding into the kinked structure. Binding of proteins including the L7Ae family can induce the formation of the kinked geometry, raising the question of whether this occurs by passive selection of the kinked structure, or a more active process in which the protein manipulates the RNA structure. We have devised a single-molecule experiment whereby immobilized L7Ae protein binds Cy3-Cy5-labeled RNA from free solution. We find that all bound RNA is in the kinked geometry, with no evidence for transitions to an extended form at the millisecond timescale of the camera. Furthermore, real-time binding experiments provide no evidence for a more extended intermediate even at the earliest times, at a time resolution of 16 ms. The data support a passive conformational selection model by which the protein selects a fraction of RNA that is already in the kinked conformation, thereby drawing the equilibrium into this form.