RESUMO
The folding of RNA into a wide range of structures is essential for its diverse biological functions from enzymatic catalysis to ligand binding and gene regulation. The unfolding and refolding of individual RNA molecules can be probed by single-molecule force spectroscopy (SMFS), enabling detailed characterization of the conformational dynamics of the molecule as well as the free-energy landscape underlying folding. Historically, high-precision SMFS studies of RNA have been limited to custom-built optical traps. Although commercial atomic force microscopes (AFMs) are widely deployed and offer significant advantages in ease-of-use over custom-built optical traps, traditional AFM-based SMFS lacks the sensitivity and stability to characterize individual RNA molecules precisely. Here, we developed a high-precision SMFS assay to study RNA folding using a commercial AFM and applied it to characterize a small RNA hairpin from HIV that plays a key role in stimulating programmed ribosomal frameshifting. We achieved rapid data acquisition in a dynamic assay, unfolding and then refolding the same individual hairpin more than 1,100 times in 15 min. In comparison to measurements using optical traps, our AFM-based assay featured a stiffer force probe and a less compliant construct, providing a complementary measurement regime that dramatically accelerated equilibrium folding dynamics. Not only did kinetic analysis of equilibrium trajectories of the HIV RNA hairpin yield the traditional parameters used to characterize folding by SMFS (zero-force rate constants and distances to the transition state), but we also reconstructed the full 1D projection of the folding free-energy landscape comparable to state-of-the-art studies using dual-beam optical traps, a first for this RNA hairpin and AFM studies of nucleic acids in general. Looking forward, we anticipate that the ease-of-use of our high-precision assay implemented on a commercial AFM will accelerate studying folding of diverse nucleic acid structures.
Assuntos
HIV/ultraestrutura , Nanotecnologia , Conformação de Ácido Nucleico , RNA Viral/ultraestrutura , HIV/química , Humanos , Microscopia de Força Atômica , Pinças Ópticas , RNA Viral/química , Imagem Individual de MoléculaRESUMO
Quantifying the energy landscape underlying protein-ligand interactions leads to an enhanced understanding of molecular recognition. A powerful yet accessible single-molecule technique is atomic force microscopy (AFM)-based force spectroscopy, which generally yields the zero-force dissociation rate constant (koff ) and the distance to the transition state (Δx≠ ). Here, we introduce an enhanced AFM assay and apply it to probe the computationally designed protein DIG10.3 binding to its target ligand, digoxigenin. Enhanced data quality enabled an analysis that yielded the height of the transition state (ΔG≠ =6.3±0.2â kcal mol-1 ) and the shape of the energy barrier at the transition state (linear-cubic) in addition to the traditional parameters [koff (=4±0.1×10-4 â s-1 ) and Δx≠ (=8.3±0.1â Å)]. We expect this automated and relatively rapid assay to provide a more complete energy landscape description of protein-ligand interactions and, more broadly, the diverse systems studied by AFM-based force spectroscopy.
Assuntos
Desenho Assistido por Computador , Digoxigenina/química , Proteínas/química , Termodinâmica , Sítios de Ligação , Ligantes , Microscopia de Força AtômicaRESUMO
Single-molecule force spectroscopy (SMFS) is a powerful technique to characterize the energy landscape of individual proteins, the mechanical properties of nucleic acids, and the strength of receptor-ligand interactions. Atomic force microscopy (AFM)-based SMFS benefits from ongoing progress in improving the precision and stability of cantilevers and the AFM itself. Underappreciated is that the accuracy of such AFM studies remains hindered by inadvertently stretching molecules at an angle while measuring only the vertical component of the force and extension, degrading both measurements. This inaccuracy is particularly problematic in AFM studies using double-stranded DNA and RNA due to their large persistence length (p ≈ 50 nm), often limiting such studies to other SMFS platforms (e.g., custom-built optical and magnetic tweezers). Here, we developed an automated algorithm that aligns the AFM tip above the DNA's attachment point to a coverslip. Importantly, this algorithm was performed at low force (10-20 pN) and relatively fast (15-25 s), preserving the connection between the tip and the target molecule. Our data revealed large uncorrected lateral offsets for 100 and 650 nm DNA molecules [24 ± 18 nm (mean ± standard deviation) and 180 ± 110 nm, respectively]. Correcting this offset yielded a 3-fold improvement in accuracy and precision when characterizing DNA's overstretching transition. We also demonstrated high throughput by acquiring 88 geometrically corrected force-extension curves of a single individual 100 nm DNA molecule in â¼40 min and versatility by aligning polyprotein- and PEG-based protein-ligand assays. Importantly, our software-based algorithm was implemented on a commercial AFM, so it can be broadly adopted. More generally, this work illustrates how to enhance AFM-based SMFS by developing more sophisticated data-acquisition protocols.
RESUMO
Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize mechanical properties of biomolecules. Historically, accessibility relies upon the nonspecific adhesion of biomolecules to a surface and a cantilever and, for proteins, the integration of the target protein into a polyprotein. However, this assay results in a low yield of high-quality data, defined as the complete unfolding of the polyprotein. Additionally, nonspecific surface adhesion hinders studies of α-helical proteins, which unfold at low forces and low extensions. Here, we overcame these limitations by merging two developments: (i) a polyprotein with versatile, genetically encoded short peptide tags functionalized via a mechanically robust Hydrazino-Pictet-Spengler ligation and (ii) the efficient site-specific conjugation of biomolecules to PEG-coated surfaces. Heterobifunctional anchoring of this polyprotein construct and DNA via copper-free click chemistry to PEG-coated substrates and a strong but reversible streptavidin-biotin linkage to PEG-coated AFM tips enhanced data quality and throughput. For example, we achieved a 75-fold increase in the yield of high-quality data and repeatedly probed the same individual polyprotein to deduce its dynamic force spectrum in just 2 h. The broader utility of this polyprotein was demonstrated by measuring three diverse target proteins: an α-helical protein (calmodulin), a protein with internal cysteines (rubredoxin), and a computationally designed three-helix bundle (α3D). Indeed, at low loading rates, α3D represents the most mechanically labile protein yet characterized by AFM. Such efficient SMFS studies on a commercial AFM enable the rapid characterization of macromolecular folding over a broader range of proteins and a wider array of experimental conditions (pH, temperature, denaturants). Further, by integrating these enhancements with optical traps, we demonstrate how efficient bioconjugation to otherwise nonstick surfaces can benefit diverse single-molecule studies.
Assuntos
Proteínas/química , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Conformação Proteica em alfa-Hélice , TemperaturaRESUMO
Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is widely used to mechanically measure the folding and unfolding of proteins. However, the temporal resolution of a standard commercial cantilever is 50-1000 µs, masking rapid transitions and short-lived intermediates. Recently, SMFS with 0.7-µs temporal resolution was achieved using an ultrashort (L = 9 µm) cantilever on a custom-built, high-speed AFM. By micromachining such cantilevers with a focused ion beam, we optimized them for SMFS rather than tapping-mode imaging. To enhance usability and throughput, we detected the modified cantilevers on a commercial AFM retrofitted with a detection laser system featuring a 3-µm circular spot size. Moreover, individual cantilevers were reused over multiple days. The improved capabilities of the modified cantilevers for SMFS were showcased by unfolding a polyprotein, a popular biophysical assay. Specifically, these cantilevers maintained a 1-µs response time while eliminating cantilever ringing (Q â 0.5). We therefore expect such cantilevers, along with the instrumentational improvements to detect them on a commercial AFM, to accelerate high-precision AFM-based SMFS studies.
Assuntos
Microscopia de Força Atômica/métodos , Análise Espectral/métodosRESUMO
Advanced optical traps can probe single molecules with Ångstrom-scale precision, but drift limits the utility of these instruments. To achieve Å-scale stability, a differential measurement scheme between a pair of laser foci was introduced that substantially exceeds the inherent mechanical stability of various types of microscopes at room temperature. By using lock-in detection to measure both lasers with a single quadrant photodiode, we enhanced the differential stability of this optical reference frame and thereby stabilized an optical-trapping microscope to 0.2 Å laterally over 100 s based on the Allan deviation. In three dimensions, we achieved stabilities of 1 Å over 1,000 s and 1 nm over 15 h. This stability was complemented by high measurement bandwidth (100 kHz). Overall, our compact back-scattered detection enables an ultrastable measurement platform compatible with optical traps, atomic force microscopy, and optical microscopy, including super-resolution techniques.
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While traditional models of protein adsorption focus primarily on direct protein-surface interactions, recent findings suggest that protein-protein interactions may play a central role. Using high-throughput intermolecular resonance energy transfer (RET) tracking, we directly observed dynamic, protein-protein associations of bovine serum albumin on polyethylene glycol modified surfaces. The associations were heterogeneous and reversible, and associating molecules resided on the surface for longer times. The appearance of three distinct RET states suggested a spatially heterogeneous surface - with areas of high protein density (i.e., strongly interacting clusters) coexisting with mobile monomers. Distinct association states exhibited characteristic behavior, i.e., partial-RET (monomer-monomer) associations were shorter-lived than complete-RET (protein-cluster) associations. While the fractional surface area covered by regions with high protein density (i.e., clusters) increased with increasing concentration, the distribution of contact times between monomers and clusters was independent of solution concentration, suggesting that associations were a local phenomenon, and independent of the global surface coverage.
Assuntos
Domínios e Motivos de Interação entre Proteínas/fisiologia , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Animais , Bovinos , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Estrutura Terciária de Proteína , Propriedades de SuperfícieRESUMO
The interactions between adsorbate molecules and hydrophobic surfaces are of significant interest due to their importance in a variety of biological and separation processes. However, it is challenging to extrapolate macroscopic ensemble-averaged force measurements to molecular-level phenomena. Using total internal reflection fluorescence microscopy to image individual molecules at hydrophobic solid-aqueous interfaces, we directly observed dynamic behavior associated with the interactions between fluorescently labeled dodecanoic acid (our probe molecules) and self-assembled monolayers (SAM) comprising n-alkyltriethoxysilanes with systematically increasing chain length (from n = 4-18). In all cases, we observed at least two characteristic surface residence times and two diffusive modes, suggesting the presence of multiple distinct adsorbed populations. In general, the mean surface residence time increased and the mobility decreased with increasing SAM chain length, consistent with stronger probe-surface interactions. However, these trends were not primarily due to changes in characteristic residence times or diffusion coefficients associated with the individual populations but rather to a dramatic increase in the fraction associated with the long-lived slow-moving population(s) on long-chain SAMs. In particular, on longer (16-18 carbon) alkylsilane monolayers, the probe molecule exhibited far fewer desorption-mediated "flights" than on short (4-6 carbon) monolayers. Additionally, probes on the longer chain surfaces were much more likely to exhibit extended surface residence times as opposed to short transient surface visits.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Ácidos Láuricos/química , Simulação de Dinâmica Molecular , Silanos/química , Alcanos/química , Difusão , Corantes Fluorescentes/química , Propriedades de Superfície , Água/químicaRESUMO
The "soft" (i.e., noncovalent) interactions between molecules and surfaces are complex and highly varied (e.g., hydrophobic, hydrogen bonding, and ionic), often leading to heterogeneous interfacial behavior. Heterogeneity can arise either from the spatial variation of the surface/interface itself or from molecular configurations (i.e., conformation, orientation, aggregation state, etc.). By observing the adsorption, diffusion, and desorption of individual fluorescent molecules, single-molecule tracking can characterize these types of heterogeneous interfacial behavior in ways that are inaccessible to traditional ensemble-averaged methods. Moreover, the fluorescence intensity or emission wavelength (in resonance energy transfer experiments) can be used to track the molecular configuration and simultaneously directly relate this to the resulting interfacial mobility or affinity. In this feature article, we review recent advances involving the use of single-molecule tracking to characterize heterogeneous molecule-surface interactions including multiple modes of diffusion and desorption associated with both internal and external molecular configuration, Arrhenius-activated interfacial transport, spatially dependent interactions, and many more.
Assuntos
Espectrometria de Fluorescência/métodos , Humanos , Conformação Molecular , Óleos/química , Proteínas/química , Tensoativos/química , Água/químicaRESUMO
High throughput single molecule tracking methods were developed to perform quantitative analyses of rare molecular populations. An optimization strategy for single molecule tracking at interfaces is described that allowed tracking of ~10(6) unique trajectories. These large statistical datasets were analyzed in order to identify and characterize distinct molecular populations based on their characteristic dynamic behavior (residence time or surface diffusion) and/or their spatial distribution. Cumulative (i.e. integrated) probability distributions were found to be several orders of magnitude more sensitive to rare populations than were raw probability distributions. Mapping using Accumulated Probe Trajectories (MAPT) was used to characterize molecular populations associated with rare surface heterogeneities. Importantly, large sample sizes were found to result in a dramatic enhancement in the ability to identify rare populations and to resolve their dynamic and spatial parameters.
Assuntos
Probabilidade , Algoritmos , Cromatografia Líquida de Alta PressãoRESUMO
The surface characterization of 'soft' materials presents a significant scientific challenge, particularly under 'wet' in situ conditions where a wide variety of non-covalent interactions may be relevant. Here we introduce a new chemical imaging method, MAPT (mapping using accumulated probe trajectories) that generates images of surface interactions by distributing different aspects of molecular probe trajectories into distinct locations and then combining many trajectories to generate spatial maps. The maps are super-resolution in nature, because they are accumulated from highly localized single-molecule observations. Unlike other super-resolution techniques, which report only photon or point counts, our analysis generates spatial maps of physical quantities (adsorption rate, desorption probability, local surface diffusion coefficient, surface coverage/occupancy) that are directly associated with the molecular interactions between the probe molecule and the surface. We demonstrate the feasibility of this characterization using a surface patterned with various degrees of hydrophobicity.
RESUMO
By directly observing molecular trajectories on a chemically heterogeneous surface, we have identified two distinct modes of diffusion involving (1) displacements within isolated surface islands (crawling mode), and (2) displacements where a molecule desorbs from an island, diffuses through the adjacent liquid phase, and readsorbs on another island (flying mode). The diffusion coefficients corresponding to these two modes differ by an order of magnitude, and both modes are also observed on chemically homogeneous surfaces. Comparison with previous results suggested that desorption-mediated diffusion is the primary transport mechanism in self-assembled monolayer formation.
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Fluorescence recovery after photobleaching was used to characterize the diffusion of fluorescently labeled phospholipids at the oil/water interface for oil viscosities that varied over four orders of magnitude. Measurements were performed over a range of surface concentrations corresponding to molecular areas of 40-130 A(2)/molecule. As expected, the interfacial diffusion coefficient increased with molecular area, saturating at an area of approximately 100 A(2)/molecule. At molecular areas below approximately 80 A(2)/molecule, macroscopic domains of a condensed monolayer phase were observed; the diffusion of these domains was characterized by direct tracking and trajectory analysis. For oils with viscosity =1500 cP, the diffusion coefficients of both individual probe molecules and condensed domains were consistent with a mechanism where the objects moved within the interface, experiencing drag from the adjacent bulk phases. Because this drag was dominated by the oil viscosity, the diffusion coefficients decreased proportionally to the inverse of the oil viscosity. However, for oils with higher viscosity, the diffusion coefficient of individual probe molecules decreased much more slowly. These observations suggested that two diffusive mechanisms are involved: one where surfactant molecules move within the interface and one that is analogous to the activated "hopping" processes that occur at the solid/liquid interface. This latter mode becomes significant only for very viscous oil phases.
Assuntos
Óleos/química , Fosfolipídeos/química , Água/química , Difusão , Fotodegradação , Propriedades de Superfície , ViscosidadeRESUMO
Single-molecule total internal reflectance fluorescence microscopy was used to observe the dynamic behavior of >4000 bovine serum albumin objects at the silicone oil-water interface. The surface residence time distribution indicated the presence of three populations at the interface. Each population had a characteristic fluorescence intensity and distinctive interfacial diffusion behavior. Larger fluorescence intensity correlated with longer residence times and slower diffusion. These combined observations of fluorescence intensity, surface residence time, and interfacial diffusion suggested that the three populations represent monomers, dimers, and trimers respectively.
Assuntos
Óleos/química , Soroalbumina Bovina/química , Água/química , Animais , Bovinos , Espectrometria de Fluorescência , Propriedades de SuperfícieRESUMO
Using total internal reflection fluorescence microscopy (TIRFM), we have observed the directed motion of 20 nm probe particles on specific regions of surfaces that exhibited strong gradients of hydrophobicity. Patterned surfaces were prepared by selective photodegradation (using a contact photomask) of a hydrophobically modified fused silica surface. The lateral distribution of hydrophobicity was characterized in situ using the selective affinity of amphiphilic probes (i.e., hydrophobic interaction microscopy). Probe particles were observed to move unidirectionally from regions of lower to higher to hydrophobicity over distances of approximately 1 microm when the hydrophobicity gradient was greater than d(cos theta)/dx = 0.05 +/- 0.02 microm(-1), where theta is the water contact angle on the bare surface. Only adsorption events were observed on energetically homogeneous surface regions.
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A Langmuir monolayer trough that is equipped for simultaneous microrheology and standard rheology measurements has been constructed. The central elements are the trough itself with a full range of optical tools accessing the air-water interface from below the trough and a portable knife-edge torsion pendulum that can access the interface from above. The ability to simultaneously measure the mechanical response of Langmuir monolayers on very different length scales is an important step for our understanding of the mechanical response of two-dimensional viscoelastic networks.
Assuntos
Bicamadas Lipídicas/química , Reologia/instrumentação , Lipossomas Unilamelares/química , Reologia/métodos , ViscosidadeRESUMO
We report on the surface rheology of cross-linked F-actin networks associated with a lipid monolayer at the air-water interface of a Langmuir monolayer. The rheological measurements are made using a Couette cell. These data demonstrate that the network has a finite elastic modulus that grows as a function of the cross-linking concentration. We also note that under steady-state flow the system behaves as a power-law fluid in which the effective viscosity decreases with imposed shear.