RESUMO
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.
Assuntos
Pinças Ópticas , Proteínas , Fenômenos Biofísicos , Comunicação Celular , Cinética , Proteínas/químicaRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Saxitoxina/química , Simulação de Dinâmica Molecular , Nanotecnologia/métodos , Neurotoxinas/química , Pinças ÓpticasRESUMO
Thy-1 and αvß3 integrin mediate bidirectional cell-to-cell communication between neurons and astrocytes. Thy-1/αvß3 interactions stimulate astrocyte migration and the retraction of neuronal prolongations, both processes in which internal forces are generated affecting the bimolecular interactions that maintain cell-cell adhesion. Nonetheless, how the Thy-1/αvß3 interactions respond to mechanical cues is an unresolved issue. In this study, optical tweezers were used as a single-molecule force transducer, and the Dudko-Hummer-Szabo model was applied to calculate the kinetic parameters of Thy-1/αvß3 dissociation. A novel experimental strategy was implemented to analyze the interaction of Thy-1-Fc with nonpurified αvß3-Fc integrin, whereby nonspecific rupture events were corrected by using a new mathematical approach. This methodology permitted accurately estimating specific rupture forces for Thy-1-Fc/αvß3-Fc dissociation and calculating the kinetic and transition state parameters. Force exponentially accelerated Thy-1/αvß3 dissociation, indicating slip bond behavior. Importantly, nonspecific interactions were detected even for purified proteins, highlighting the importance of correcting for such interactions. In conclusion, we describe a new strategy to characterize the response of bimolecular interactions to forces even in the presence of nonspecific binding events. By defining how force regulates Thy-1/αvß3 integrin binding, we provide an initial step towards understanding how the neuron-astrocyte pair senses and responds to mechanical cues.