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Determining the non-specific and specific electrostatic contributions of magnesium binding to RNA is a challenging problem. We introduce a single-molecule method based on measuring the folding energy of a native RNA in magnesium and at its equivalent sodium concentration. The latter is defined so that the folding energy in sodium equals the non-specific electrostatic contribution in magnesium. The sodium equivalent can be estimated according to the empirical 100/1 rule (1 M NaCl is equivalent to 10 mM MgCl

Domain swapping is a mechanism of protein oligomerization by which two or more subunits exchange structural elements to generate an intertwined complex. Numerous studies support a diversity of swapping mechanisms in which structural elements can be exchanged at different stages of the folding pathway of a subunit. Here, we used single-molecule optical tweezers technique to analyze the swapping mechanism of the forkhead DNA-binding domain of human transcription factor FoxP1. FoxP1 populates folded monomers in equilibrium with a swapped dimer. We generated a fusion protein linking two FoxP1 domains in tandem to obtain repetitive mechanical folding and unfolding trajectories. Thus, by stretching the same molecule several times, we detected either the independent folding of each domain or the elusive swapping step between domains. We found that a swapped dimer can be formed directly from fully or mostly folded monomer. In this situation, the interaction between the monomers in route to the domain-swapped dimer is the rate-limiting step. This approach is a useful strategy to test the different proposed domain swapping mechanisms for proteins with relevant physiological functions.

Impaired mucociliary clearance (MCC) is a key feature of many airway diseases, including asthma, bronchiectasis, chronic obstructive pulmonary disease, cystic fibrosis, and primary ciliary dyskinesia. To improve MCC and develop new treatments for these diseases requires a thorough understanding of how mucus concentration, mucus composition, and ciliary activity affect MCC, and how different therapeutics impact this process. Although differentiated cultures of human airway epithelial cells are useful for investigations of MCC, the extent of ciliary coordination in these cultures varies, and the mechanisms controlling ciliary orientation are not completely understood. By introducing a pattern of ridges and grooves into the underlying collagen substrate, we demonstrate for the first time, to our knowledge, that changes in the extracellular matrix can induce ciliary alignment. Remarkably, 90% of human airway epithelial cultures achieved continuous directional mucociliary transport (MCT) when grown on the patterned substrate. These cultures maintain transport for months, allowing carefully controlled investigations of MCC over a wide range of normal and pathological conditions. To characterize the system, we measured the transport of bovine submaxillary gland mucin (BSM) under several conditions. Transport of 5% BSM was significantly reduced compared with that of 2% BSM, and treatment of 5% BSM with the reducing agent tris(2-carboxyethyl)phosphine (TCEP) reduced viscosity and increased the rate of MCT by approximately twofold. Addition of a small amount of high-molecular-weight DNA increased mucus viscosity and reduced MCT by ∼75%, demonstrating that the composition of mucus, as well as the concentration, can have significant effects on MCT. Our results demonstrate that a simple patterning of the collagen substrate results in highly coordinated ciliated cultures that develop directional MCT, and can be used to investigate the mechanisms controlling the regulation of ciliary orientation. Furthermore, the results demonstrate that this method provides an improved system for studying the effects of mucus composition and therapeutic agents on MCC.

Thylakoid membranes in chloroplasts contain photosynthetic protein complexes that convert light energy into chemical energy. Photosynthetic protein complexes are considered to undergo structural reorganization to maintain the efficiency of photochemical reactions. A detailed description of the mobility of photosynthetic complexes in real time is necessary to understand how macromolecular organization of the membrane is altered by environmental fluctuations. Here, we used high-speed atomic force microscopy to visualize and characterize the in situ mobility of individual protein complexes in grana thylakoid membranes isolated from Spinacia oleracea. Our observations reveal that these membranes can harbor complexes with at least two distinctive classes of mobility. A large fraction of grana membranes contained proteins with quasistatic mobility exhibiting molecular displacements smaller than 10 nm2. In the remaining fraction, the protein mobility is variable with molecular displacements of up to 100 nm2. This visualization at high spatiotemporal resolution enabled us to estimate an average diffusion coefficient of ∼1 nm2 s-1. Interestingly, both confined and Brownian diffusion models could describe the protein mobility of the second group of membranes. We also provide the first direct evidence, to our knowledge, of rotational diffusion of photosynthetic complexes. The rotational diffusion of photosynthetic complexes could be an adaptive response to the high protein density in the membrane to guarantee the efficiency of electron transfer reactions. This characterization of the mobility of individual photosynthetic complexes in grana membranes establishes a foundation that could be adapted to study the dynamics of the complexes inside intact and photosynthetically functional thylakoid membranes to be able to understand its structural responses to diverse environmental fluctuations.

ATP-dependent proteases are crucial to maintain cellular protein homeostasis. Here we used single-molecule assays to characterize the mechanism by which the AAA+ protease ClpXP from E. coli transforms energy from ATP hydrolysis into mechanical work to drive protein translocation and unfolding. We establish that ATP hydrolysis and phosphate release occur in the burst phase during conformational changes of the motor, while ADP release and ATP binding happen in the dwell between bursts. We find that the residues of the highly-conserved translocating pore loops in the central ClpX pore determine the efficiency of substrate unfolding and translocation, and are crucial for the mechanochemical coupling and the power generated by ClpXP. Interestingly, we observe that the conformational resetting of the pore loops between consecutive power strokes appears to time both the dwell duration and the release of ADP. Together, our results indicate that: i) the mechanochemical coupling of the motor and its unfolding capability are mediated by the size of the residues in the translocating loops, ii) the unfolding capability of the motor does not depend on the burst size or the grip as previously proposed, but instead it depends on the power -work produced per unit time- generated by the motor, iii) provide insights into why evolution has selected a conserved sequence-motif for the translocating loops of prokaryotic and eukaryotic AAA+ proteases, and iv) indicate that ClpXP's mechanism deviates from other well studied molecular motors (such as the Phi29 DNA packaging motor).

Protein folding is often studied in the context of full-length polypeptides in solution. However, the post-translational folding pathway may not recreate the pathway of folding when a protein is being synthesized by the ribosome. The effects of the ribosome and the rate of translation on folding are not well understood, partially due to the difficulty of probing the nascent chain without also affecting the ribosome. Using optical tweezers, we can look in detail at the nascent chain at the single-molecule level with minimal perturbation to the ribosome. Previous studies have shown that the ribosomal surface can act electrostatically to slow the kinetics of folding. Those studies used a linker to vary the distance from the surface and did not look at changes in sequence availability or multidomain proteins. To further understand cotranslational folding, we are studying the folding pathway of a two-domain calcium-binding protein calerythrin. Our results show that the full-length protein in solution folds robustly through a C-domain intermediate, but truncated versions of the protein can fold to an N domain or a misfolded state. During translation, the C-terminal residues required for native folding are not yet available, but the other observed intermediates could fold, in principle. By observing the folding at various sequence positions, and thus nascent chain lengths, we found that the ribosome can not only slow folding, but can also affect the unfolding rates, particularly increasing the unfolding rate of the misfolded state. The ribosome thus prevents the formation of this unproductive state via two mechanisms: decreasing the probability of folding and decreasing the stability of the folded state. This study gives further insight into the importance of the ribosome to regulate protein structure, and opens up new questions about the interplay between elongation and folding.

By exerting mechanical force it is possible to unfold/refold RNA molecules one at a time. In a small range of forces, an RNA molecule can hop between the folded and the unfolded state with force-dependent kinetic rates. Here, we introduce a mesoscopic model to analyze the hopping kinetics of RNA hairpins in an optical tweezers setup. The model includes different elements of the experimental setup (beads, handles and RNA sequence) and limitations of the instrument (time lag of the force-feedback mechanism and finite bandwidth of data acquisition). We investigated the influence of the instrument on the measured hopping rates. Results from the model are in good agreement with the experiments reported in the companion article (1). The comparison between theory and experiments allowed us to infer the values of the intrinsic molecular rates of the RNA hairpin alone and to search for the optimal experimental conditions to do the measurements. We conclude that long handles and soft laser traps represent the best conditions to extract rate estimates that are closest to the intrinsic molecular rates. The methodology and rationale presented here can be applied to other experimental setups and other molecules., Comment: PDF file, 32 pages including 9 figures plus supplementary material

Mechanical unfolding and refolding of single RNA molecules have previously been observed in optical traps as sudden changes in molecular extension. Two methods have been traditionally used: “force-ramp”, with the applied force continuously changing, and “hopping”. In hopping experiments the force is held constant and the molecule jumps spontaneously between two different states. Unfolding/refolding rates are measured directly, but only over a very narrow range of forces. We have now developed a force-jump method to measure the unfolding and refolding rates independently over a wider range of forces. In this method, the applied force is rapidly stepped to a new value and either the unfolding or refolding event is monitored through changes in the molecular extension. The force-jump technique is compared to the force-ramp and hopping methods by using a 52-nucleotide RNA hairpin with a three-nucleotide bulge, i.e., the transactivation response region RNA from the human immunodeficiency virus. We find the unfolding kinetics and Gibbs free energies obtained from all three methods to be in good agreement. The transactivation response region RNA hairpin unfolds in an all-or-none two-state reaction at any loading rate with the force-ramp method. The unfolding reaction is reversible at small loading rates, but shows hysteresis at higher loading rates. Although the RNA unfolds and refolds without detectable intermediates in constant-force conditions (hopping and force-jump), it shows partially folded intermediates in force-ramp experiments at higher unloading rates. Thus, we find that folding of RNA hairpins can be more complex than a simple single-step reaction, and that application of several methods can improve understanding of reaction mechanisms.

Measurements made on large ensembles of molecules are routinely interpreted using thermodynamics, but the normal rules of thermodynamics may not apply to measurements made on single molecules. Using a polymer stretching experiment as an example, it is shown that in the limit of a single, short molecule the outcome of experimental measurements may depend on which variables are held fixed and which are allowed to fluctuate. Thus an experiment in which the end-to-end distance of the polymer molecule is fixed and the tension fluctuates yields a different result than an experiment where the force is fixed and the end-to-end distance fluctuates. It is further shown that this difference is due to asymmetry in the distribution of end-to-end distances for a single molecule, and that the difference vanishes in the appropriate thermodynamic limit; that is, as the polymer molecule becomes long compared to its persistence length. Despite these differences, much of the thermodynamic formalism still applies on the single-molecule level if the thermodynamic free energies are replaced with appropriate potentials of mean force. The primary remaining differences are consequences of the fact that unlike the free energies, the potentials of mean force are not in general homogeneous functions of their variables. The basic thermodynamic concepts of an intensive or extensive quantity, and the thermodynamic relationships that follow from them, are therefore less useful for interpreting single-molecule experiments.

Using optical tweezers in combination with confocal fluorescence microscopy in a controlled microfluidics environment, results in a robust and versatile methodology to study mechanisms occurring at all levels of the cellular metabolism, up to cell membrane interactions and beyond. Both the high temporal and spatial resolutions that correlative optical tweezers-fluorescence microscopy (CTFM) offers represent an asset to investigate processes with short lifetimes. Additionally, by being able to manipulate and exert tensions to selected molecules, we gain access to important structural features. To demonstrate the potential of this hybrid technique, we performed a series of experiments to study the folding/unfolding dynamics of an intracellular signaling protein and the packaging of DNA within a bacteriophage capsid. Both model systems present transient states with fast kinetics, which would be overlooked using other techniques. Our results show that the applications of this single‑molecule technology are not limited to the field of nucleic acids research and quickly advance to new venues.

During transcription elongation, E. coli RNA polymerase (RNAP) alternates between periods of processive nucleotide addition and periods of pausing. Various mechanisms promote pausing. In particular, increased pausing at a “consensus pause element” of the form G-10Y-1G+1 (where Y-1 corresponds to the 3′ RNA end) has been described (Science 344:1042, 2014; Science 344:1285, 2014). Such pause sites have been shown to exhibit increased efficiency in the RNAP βD446A mutant; this effect can be understood from the interaction between WT RNAP βD446 and G+1, which stabilizes RNAP in a post-translocated register in the presence of a G+1, and thus decreases pausing (Science 344:1285, 2014).In this work, we use high-resolution optical tweezers to characterize the dynamics of both processive nucleotide elongation and pausing by WT and βD446A RNAP at very high spatial and temporal resolutions, under various force conditions. Our ability to resolve sub-second pausing events provide new insights into the dynamics of RNAP translocation and pausing during elongation, and their modulation by sequence-specific RNAP-DNA interactions.

Single walled carbon nanotubes (SWNTs) are one of the promising nanomaterials for various applications in nanotechnology. In biomedical fields, hybrids of DNA and SWNT (DNA-SWNT hybrids) are expected to be attractive nanodevices for drug delivery and DNA sensors. For preparing DNA-SWNT hybrids, mixture of SWNT and DNA solutions are prepared via sonication. Although DNA-SWNT hybridization techniques have been established, the diverse sonication conditions in every paper may affect structures of DNA-SWNT hybrids. Here we report a method of monitoring adsorption process of DNA to SWNT surfaces using fluorescent labeled single-stranded DNA (Fluor-ssDNA). It is known that fluorescent emission of fluorescent dyes is quenched when the dye is adsorbed on SWNT surfaces. Using this unique quenching phenomenon, we evaluated adsorption process of Fluor-ssDNA on SWNT surfaces. In the experiments, 0.25 mg of SWNT powder, 50 uL of Fluor-ssDNA solution (1.0 mg/mL in tris (hydroxymethyl aminomethane buffer solution, 200mM, pH 7.5)), and 450 uL of the buffer were mixed, and then sonicated under three different sonication conditions as follows: probe type with amplitude 60% of 130 W (P60), probe type with amplitude 20% of 130 W (P20), and bath type with 80 W (B80). In order to monitor the adsorption process, samples were characterized by fluorescent spectroscopy and atomic force microscopy. In the case of P60, fluorescent intensity of Fluor-ssDNA decreased 98% within 2 min. This suggests that most of Fluor-ssDNA rapidly adsorbed on SWNT surfaces. However, AFM images demonstrated that it took more time to form mono-dispersed DNA-SWNT hybrids. In the case of P20 and B80, 30 min was needed for 98% quenching. In addition, reproducibility of the B80 data was much higher than that of P20. This is an advantage of bath type sonicator.

Sulfo-cerebrosides (SCB) are glycosphingolipids found in high concentrations in the central nervous system (CNS), particularly in myelin where they play an important structural role. In addition, these lipids play an important role in various biological processes such as establishing a connection between the neuronal axon and myelin, and regulating cell growth and neuronal plasticity. Moreover, recent in-vitro studies from our group have shown that astrocytes avidly endocyte liposomes containing SCB without inducing a pro-inflammatory response. This indicates that astrocytes are involved in the homeostasis of the SCB in the CNS. In this work, we explore the specificity of the process of endocytosis of liposomes containing SCB, using an astrocytoma cell line as a model. Our hypothesis is that this process is receptor-mediated and not due to an electrostatic interaction with the cell surface. We show that (PG enriched) negatively charged liposomes do enhance endocytosis, but at a significantly lower level compared to SCB. We also show that specific uptake can be inhibited by screening the surface with polyethylene glycol. Additionally, we show through the use of endocytotic inhibitors that the SCB endocytotic route is a clatherin dependent route, which differs from non-specific liposome uptake routes. In these experiments, cells were cultured with liposomes labeled with the membrane fluorescent probe (Texas Red) to measure internalization, and a liposome content marker (calcein) to measure release. Measurements were carried out by flow cytometry and the results confirmed by fluorescence microscopy. Since low density lipoproteins (LDL) have been suggested to participate in SCB transport, we propose that the endocytotic pathway involves the LDL receptor (LDLr). Preliminary results show that SCB endocytosis is inhibited by LDLr specific antibodies.

Nucleic acids and proteins are increasingly being analyzed using solid-state nanopores. The nanopore method relies on the measurement of transient conductance changes of the nanopores as charged biomolecules are being electrophoretically driven into it. Although electrical detection is highly appealing for the label-free analysis of biological samples, our understanding of the dynamics of charged biopolymers through such nanoconfined geometries is limited by the information captured by the electrical signal alone.We report the development of an instrument capable of interrogating solid-state nanopore devices by simultaneous electrical and optical measurements. Instead of the conventional TEM-drilled nanopores, we used a method pioneered by our group, and fabricate nanopores in a thin SiNx membrane via dielectric breakdown in solution . In this presentation, we show how a range of odd behaviors of the ionic current can be explained with parallel fluorescence imaging of the nanopore. Our custom instrument allows us to: (i) study these anomalous events; (ii) probe the dynamics of DNA capture; (iii) monitor, for the first time, the creation process of a nanopore; and (iv) determine its location on the dielectric membrane;.A better understanding of these anomalous single-molecule events during DNA passage through solid-state nanopores is essential to increase reliability of nanopore-based sensing.