Characterization of the interaction between the Sec61 translocon complex and ppαF using optical tweezers.

The Sec61 translocon allows the translocation of secretory preproteins from the cytosol to the endoplasmic reticulum lumen during polypeptide biosynthesis. These proteins possess an N-terminal signal peptide (SP) which docks at the translocon. SP mutations can abolish translocation and cause diseases, suggesting an essential role for this SP/Sec61 interaction. However, a detailed biophysical characterization of this binding is still missing. Here, optical tweezers force spectroscopy was used to characterize the kinetic parameters of the dissociation process between Sec61 and the SP of prepro-alpha-factor. The unbinding parameters including off-rate constant and distance to the transition state were obtained by fitting rupture force data to Dudko-Hummer-Szabo models. Interestingly, the translocation inhibitor mycolactone increases the off-rate and accelerates the SP/Sec61 dissociation, while also weakening the interaction. Whereas the translocation deficient mutant containing a single point mutation in the SP abolished the specificity of the SP/Sec61 binding, resulting in an unstable interaction. In conclusion, we characterize quantitatively the dissociation process between the signal peptide and the translocon, and how the unbinding parameters are modified by a translocation inhibitor.

Determination of protein-protein interactions at the single-molecule level using optical tweezers.

Biomolecular interactions are at the base of all physical processes within living organisms; the study of these interactions has led to the development of a plethora of different methods. Among these, single-molecule (in singulo) experiments have become relevant in recent years because these studies can give insight into mechanisms and interactions that are hidden for ensemble-based (in multiplo) methods. The focus of this review is on optical tweezer (OT) experiments, which can be used to apply and measure mechanical forces in molecular systems. OTs are based on optical trapping, where a laser is used to exert a force on a dielectric bead; and optically trap the bead at a controllable position in all three dimensions. Different experimental approaches have been developed to study protein­protein interactions using OTs, such as: (1) refolding and unfolding in trans interaction where one protein is tethered between the beads and the other protein is in the solution; (2) constant force in cis interaction where each protein is bound to a bead, and the tension is suddenly increased. The interaction may break after some time, giving information about the lifetime of the binding at that tension. And (3) force ramp in cis interaction where each protein is attached to a bead and a ramp force is applied until the interaction breaks. With these experiments, parameters such as kinetic constants (koff, kon), affinity values (KD), energy to the transition state ΔG≠, distance to the transition state Δx≠ can be obtained. These parameters characterize the energy landscape of the interaction. Some parameters such as distance to the transition state can only be obtained from force spectroscopy experiments such as those described here.

New Insights into the Mechanism of Action of the Drug Chloroquine: Direct Interaction with DNA and Cytotoxicity.

Chloroquine (CLQ) and hydroxychloroquine (HCLQ) are compounds largely employed in the treatment of various human diseases for decades. Nevertheless, a number of intrinsic details concerning their mechanisms of action, especially at the molecular level, are still unknown or have presented controversial results in the literature. Using optical tweezers, here, we investigate at the single-molecule level the molecular mechanism of action of the drug CLQ in its intrinsic interaction with the double-stranded (ds)DNA molecule, one of its targets inside cells, determining the binding modes and the physicochemical (binding) parameters of the interaction. In particular, we show that the ionic strength of the surrounding medium strongly influences such interaction, changing even the main binding mode. In addition, the cytotoxicity of CLQ against three different cell lines was also investigated here, allowing one to evaluate and compare the effect of the drug on the cell viability. In particular, we show that CLQ is highly cytotoxic at a very low (a few micromolar) concentration range for all cell lines tested. These results were rigorously compared to the equivalent ones obtained for the closely related compound hydroxychloroquine (HCLQ), allowing a critical comparison between the action of these drugs at the molecular and cellular levels.

Single-molecule optical tweezers reveals folding steps of the domain swapping mechanism of a protein.

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.

Protocol to measure the membrane tension and bending modulus of cells using optical tweezers and scanning electron microscopy.

The elastic properties of cell membranes, particularly the membrane tension and bending modulus, are known to be key regulators of cellular functions. Here, we present a correlative and integrated tool based on optical tweezers and scanning electron microscopy to accurately determine these properties in a variety of cell types. Although there are intrinsic difficulties associated with correlative experiments, we believe that the methods presented can be considered a suitable protocol for determining the elastic properties of cell membranes. For complete details on the use and execution of this protocol, please refer to Soares et al. (2020).

Hydroxychloroquine Exhibits a Strong Complex Interaction with DNA: Unraveling the Mechanism of Action.

In the past months, the use of the drug hydroxychloroquine has considerably increased in many countries, associated with a proposed treatment for the COVID-19 disease. Although there is no conclusive evidence about the efficacy of the drug for this purpose, surprisingly there are no conclusive studies in the literature concerning its mechanism of action inside cells, which is related to its interaction with nucleic acids. Here, we performed a robust characterization of the interaction between hydroxychloroquine and double-stranded DNA using single-molecule force spectroscopy and gel electrophoresis. Two different binding modes were identified, namely, minor groove binding for low drug concentrations and intercalation for high drug concentrations, and the sets of binding parameters were determined for each of these modes. Such results have unraveled in detail the molecular mechanism of action of the drug as a DNA ligand.

Detachment of Dunaliella tertiolecta Microalgae from a Glass Surface by a Near-Infrared Optical Trap.

We report on the observation of the detachment in situ and in vivo of Dunaliella tertiolecta microalgae cells from a glass surface using a 1064 nm wavelength trapping laser beam. The principal bends of both flagella of Dunaliella were seen self-adhered to either the top or bottom coverslip surfaces of a 50 µm thick chamber. When a selected attached Dunaliella was placed in the trapping site, it photoresponded to the laser beam by moving its body and flagellar tips, which eventually resulted in its detachment. The dependence of the time required for detachment on the trapping power was measured. No significant difference was found in the detachment time for cells detached from the top or bottom coverslip, indicating that the induced detachment was not due solely to the optical forces applied to the cells. After detachment, the cells remained within the optical trap. Dunaliella detached from the bottom were seen rotating about their long axis in a counterclockwise direction, while those detached from the top did not rotate. The rotation frequency and the minimal force required to escape from the trap were also measured. The average rotation frequency was found to be independent of the trapping power, and the swimming force of a cell escaping the laser trap ranged from 4 to 10 picoNewtons. Our observations provide insight into the photostimulus produced when a near-infrared trapping beam encounters a Dunaliella. The microalgae frequently absorb more light than they can actually use in photosynthesis, which could cause genetic and molecular changes. Our findings may open new research directions into the study of photomovement in species of Dunaliella and other swimming microorganisms that could eventually help to solve technological problems currently confronting biomass production. In future work, studies of the response to excess light may uncover unrecognized mechanisms of photoprotection and photoacclimation.

Dodecyltrimethylammonium bromide surfactant effects on DNA: Unraveling the competition between electrostatic and hydrophobic interactions.

We present a new study on the interaction of the DNA molecule with the surfactant dodecyltrimethylammonium bromide (DTAB), performed mainly with optical tweezers. Single-molecule force spectroscopy experiments performed in the low-force entropic regime allowed a robust characterization of the DNA-DTAB interaction, unveiling how the surfactant changes the mechanical properties of the biopolymer, the binding parameters, and the competition of the two mechanisms involved in the interaction: electrostatic attraction between the cationic surfactant heads and the negative phosphate backbone of the DNA and hydrophobic interactions between the tails of the bound DTAB molecules, which can result in DNA compaction in solution depending on the quantity of bound surfactant. Finally, force clamp experiments with magnetic tweezers and gel electrophoresis assays confirm that DTAB compacts DNA depending not only on the surfactant concentration but also on the conformation of the biopolymer in solution. The present study provides new insights on general aspects of the DNA-surfactant complexes formation, contributing to the fundamental knowledge of the physics of such interactions.

Transplatin ineffectiveness against cancer from a molecular perspective: A single-molecule force-spectroscopy study.

By performing single-molecule force spectroscopy with optical tweezers, we have characterized the interaction between the platinum-based compound transplatin and the DNA molecule, establishing a critical comparison with its isomer cisplatin. While transplatin is ineffective against tumor cells, its isomer is one of the most used drugs in current chemotherapies, and a molecular study on this difference performed at the single-molecule level was lacking until the present work. Our experiments show that transplatin binds DNA under low chloride concentrations (a situation usually found inside many cells) with an equilibrium association binding constant about four orders of magnitude lower than cisplatin. In addition, we have found that, at saturation, transplatin binds preferentially forming interstrand cross links and monoadducts, a situation very different from cisplatin, which forms preferentially intrastrand cross links. Such differences explain the ineffectiveness of transplatin in killing tumor cells. From a physical point of view, the present study advances in using the mechanical properties of the DNA molecule as sensors to evaluate the therapeutic efficiency of drugs.

Mechanical unfolding of a knotted protein unveils the kinetic and thermodynamic consequences of threading a polypeptide chain.

Knots are remarkable topological features in nature. The presence of knots in crystallographic structures of proteins have stimulated considerable research to determine the kinetic and thermodynamic consequences of threading a polypeptide chain. By mechanically manipulating MJ0366, a small single domain protein harboring a shallow trefoil knot, we allow the protein to refold from either the knotted or the unknotted denatured state to characterize the free energy profile associated to both folding pathways. By comparing the stability of the native state with reference to the knotted and unknotted denatured state we find that knotting the polypeptide chain of MJ0366 increase the folding energy barrier in a magnitude close to the energy cost of forming a knot randomly in the denatured state. These results support that a protein knot can be formed during a single cooperative step of folding but occurs at the expenses of a large increment on the free energy barrier.

Membrane Elastic Properties During Neural Precursor Cell Differentiation.

Neural precursor cells differentiate into several cell types that display distinct functions. However, little is known about how cell surface mechanics vary during the differentiation process. Here, by precisely measuring membrane tension and bending modulus, we map their variations and correlate them with changes in neural precursor cell morphology along their distinct differentiation fates. Both cells maintained in culture as neural precursors as well as those plated in neurobasal medium reveal a decrease in membrane tension over the first hours of culture followed by stabilization, with no change in bending modulus. During astrocyte differentiation, membrane tension initially decreases and then increases after 72 h, accompanied by consolidation of glial fibrillary acidic protein expression and striking actin reorganization, while bending modulus increases following observed alterations. For oligodendrocytes, the changes in membrane tension are less abrupt over the first hours, but their values subsequently decrease, correlating with a shift from oligodendrocyte marker O4 to myelin basic protein expressions and a remarkable actin reorganization, while bending modulus remains constant. Oligodendrocytes at later differentiation stages show membrane vesicles with similar membrane tension but higher bending modulus as compared to the cell surface. Altogether, our results display an entire spectrum of how membrane elastic properties are varying, thus contributing to a better understanding of neural differentiation from a mechanobiological perspective.

Evaluating viscoelastic properties and membrane electrical charges of red blood cells with optical tweezers and cationic quantum dots - applications to ß-thalassemia intermedia hemoglobinopathy.

Biomechanical and electrical properties are important to the performance and survival of red blood cells (RBCs) in the microcirculation. This study proposed and explored methodologies based on optical tweezers and cationic quantum dots (QDs) as biophotonic tools to characterize, in a complementary way, viscoelastic properties and membrane electrical charges of RBCs. The methodologies were applied to normal (HbA) and ß-thalassemia intermedia (Hbß) RBCs. The ß-thalassemia intermedia disease is a hereditary hemoglobinopathy characterized by a reduction (or absence) of ß-globin chains, which leads to α-globin chains precipitation. The apparent elasticity (µ) and membrane viscosity (ηm) of RBCs captured by optical tweezers were obtained in just a single experiment. Besides, the membrane electrical charges were evaluated by flow cytometry, exploring electrostatic interactions between cationic QDs, stabilized with cysteamine, with the negatively charged RBC surfaces. Results showed that Hbß RBCs are less elastic, have a higher ηm, and presented a reduction in membrane electrical charges, when compared to HbA RBCs. Moreover, the methodologies based on optical tweezers and QDs, here proposed, showed to be capable of providing a deeper and integrated comprehension on RBC rheological and electrical changes, resulting from diverse biological conditions, such as the ß-thalassemia intermedia hemoglobinopathy.

Structural characterization of the saxitoxin-targeting APTSTX1 aptamer using optical tweezers and molecular dynamics simulations.

Optical tweezers have enabled the exploration of picoNewton forces and dynamics in single-molecule systems such as DNA and molecular motors. In this work, we used optical tweezers to study the folding/unfolding dynamics of the APTSTX1-aptamer, a single-stranded DNA molecule with high affinity for saxitoxin (STX), a lethal neurotoxin. By measuring the transition force during (un)folding processes, we were able to characterize and distinguish the conformational changes of this aptamer in the presence of magnesium ions and toxin. This work was supported by molecular dynamics (MD) simulations to propose an unfolding mechanism of the aptamer-Mg+2 complex. Our results are a step towards the development of new aptamer-based STX sensors that are potentially cheaper and more sensitive than current alternatives.

Pixantrone anticancer drug as a DNA ligand: Depicting the mechanism of action at single molecule level.

In this work we use single molecule force spectroscopy performed with optical tweezers in order to characterize the complexes formed between the anticancer drug Pixantrone (PIX) and the DNA molecule, at two very different ionic strengths. Firstly, the changes of the mechanical properties of the DNA-PIX complexes were studied as a function of the drug concentration in the sample. Then, a quenched-disorder statistical model of ligand binding was used in order to determine the physicochemical (binding) parameters of the DNA-PIX interaction. In particular, we have found that the PIX molecular mechanism of action involves intercalation into the double helix, followed by a significant compaction of the DNA molecule due to partial neutralization of the phosphate backbone. Finally, this scenario of interaction was quantitatively compared to that found for the related drug Mitoxantrone (MTX), which binds to DNA with a considerably higher equilibrium binding constant and promotes a much stronger DNA compaction. The comparison performed between the two drugs can bring clues to the development of new (and more efficient) related compounds.

Rheological properties of cryptococcal polysaccharide change with fiber size, antibody binding and temperature.

Aim:Cryptococcus neoformans is the major agent of cryptococcosis. The main virulence factor is the polysaccharide (PS) capsule. Changes in cryptococcal PS properties have been poorly elucidated. Materials & methods: We analyzed the mechanical properties of secreted PS and intact capsules, using dynamic light scattering and optical tweezers. Results: Storage and loss moduli showed that secreted PS behaves as a viscoelastic liquid, while capsular PS behaves as a viscoelastic solid. The secreted PS remains as a viscoelastic fluid at different temperatures with thermal hysteresis after 85°C. Antibody binding altered the viscoelastic behavior of both secreted and capsular PS. Conclusion: Deciphering the mechanical aspects of these structures could reveal features that may have consequences in novel therapies against cryptococcosis.

Effects of caffeine on the structure and conformation of DNA: A force spectroscopy study.

Here, we use single molecule force spectroscopy performed with optical tweezers in order to investigate the interaction between Caffeine and the DNA molecule for various different concentrations of the alkaloid and under two distinct ionic strengths of the surrounding buffer. We were able to determine the mechanical changes induced on the double-helix structure due to Caffeine binding, the binding mode and the binding parameters of the interaction. The results obtained show that Caffeine binds to DNA by outside the double-helix with a higher affinity at lower ionic strengths. On the other hand, a considerable cooperativity was found only for sufficient high ionic strengths, suggesting that Caffeine may binding forming dimers and/or trimers along the double-helix under this condition. Finally, it was also shown that Caffeine stabilizes the DNA double-helix upon binding, preventing force-induced DNA melting.

New antineoplastic agent based on a dibenzoylmethane derivative: Cytotoxic effect and direct interaction with DNA.

Melanoma accounts for only 4% of all skin cancers but is among the most lethal cutaneous neoplasms. Dacarbazine is the drug of choice for the treatment of melanoma in Brazil through the public health system mainly because of its low cost. However, it is an alkylating agent of low specificity and elicits a therapeutic response in only 20% of cases. Other drugs available for the treatment of melanoma are expensive, and tumor cells commonly develop resistance to these drugs. The fight against melanoma demands novel, more specific drugs that are effective in killing drug-resistant tumor cells. Dibenzoylmethane (1,3-diphenylpropane-1,3-dione) derivatives are promising antitumor agents. In this study, we investigated the cytotoxic effect of 1,3-diphenyl-2-benzyl-1,3-propanedione (DPBP) on B16F10 melanoma cells as well as its direct interaction with the DNA molecule using optical tweezers. DPBP showed promising results against tumor cells and had a selectivity index of 41.94. Also, we demonstrated the ability of DPBP to interact directly with the DNA molecule. The fact that DPBP can interact with DNA in vitro allows us to hypothesize that such an interaction may also occur in vivo and, therefore, that DPBP may be an alternative to treat patients with drug-resistant melanomas. These findings can guide the development of new and more effective drugs.

Cell membrane biophysics with optical tweezers.

Membrane elastic properties play important roles in regulating cell shape, motility, division and differentiation. Here I review optical tweezer (OT) investigations of membrane surface tension and bending modulus, emphasizing didactic aspects and insights provided for cell biology. OT measurements employ membrane-attached microspheres to extract long cylindrical nanotubes named tethers. The Helfrich-Canham theory yields elastic parameters in terms of tether radius and equilibrium extraction force. It assumes initial point-like microsphere attachment and no cytoskeleton content within tethers. Experimental force-displacement curves reveal violations of those assumptions, and I discuss proposed explanations of such discrepancies, as well as recommended OT protocols. Measurements of elastic parameters for predominant cell types in the central nervous system yield correlations between their values and cell function. Micro-rheology OT experiments extend these correlations to viscoelastic parameters. The results agree with a quasi-universal phenomenological scaling law and are interpreted in terms of the soft glass rheology model. Spontaneously-generated cell nanotube protrusions are also briefly reviewed, emphasizing common features with tethers. Filopodia as well as tunneling nanotubes (TNT), which connect distant cells and allow transfers between their cytoplasms, are discussed, including OT tether pulling from TNTs which mediate communication among bacteria, even of different species. Pathogens, including bacteria, viruses and prions, opportunistically exploit TNTs for cell-to-cell transmission of infection, indicating that TNTs have an ancient evolutionary origin.

The energy cost of polypeptide knot formation and its folding consequences.

Knots are natural topologies of chains. Yet, little is known about spontaneous knot formation in a polypeptide chain-an event that can potentially impair its folding-and about the effect of a knot on the stability and folding kinetics of a protein. Here we used optical tweezers to show that the free energy cost to form a trefoil knot in the denatured state of a polypeptide chain of 120 residues is 5.8 ± 1 kcal mol-1. Monte Carlo dynamics of random chains predict this value, indicating that the free energy cost of knot formation is of entropic origin. This cost is predicted to remain above 3 kcal mol-1 for denatured proteins as large as 900 residues. Therefore, we conclude that naturally knotted proteins cannot attain their knot randomly in the unfolded state but must pay the cost of knotting through contacts along their folding landscape.

ß-Cyclodextrin polymer binding to DNA: Modulating the physicochemical parameters.

Cyclodextrins and cyclodextrins-modified molecules have interesting and appealing properties due to their capacity to host components that are normally insoluble or poorly soluble in water. In this work, we investigate the interaction of a ß-cyclodextrin polymer (poly-ß-CD) with λ-DNA. The polymers are obtained by the reaction of ß-CD with epichlorohydrin in alkaline conditions. We have used optical tweezers to characterize the changes of the mechanical properties of DNA molecules by increasing the concentration of poly-ß-CD in the sample. The physical chemistry of the interaction is then deduced from these measurements by using a recently developed quenched-disorder statistical model. It is shown that the contour length of the DNA does not change in the whole range of poly-ß-CD concentration (<300µM). On the other hand, significant alterations were observed in the persistence length that identifies two binding modes corresponding to the clustering of ∼2.6 and ∼14 polymer molecules along the DNA double helix, depending on the polymer concentration. Comparing these results with the ones obtained for monomeric ß-CD, it was observed that the concentration of CD that alters the DNA persistence length is considerably smaller when in the polymeric form. Also, the binding constant of the polymer-DNA interaction is three orders of magnitude higher than the one found for native (monomeric) ß-CD. These results show that the polymerization of the ß-CD strongly increases its binding affinity to the DNA molecule. This property can be wisely used to modulate the binding of cyclodextrins to the DNA double helix.