Use of DNA forceps to measure receptor-ligand dissociation equilibrium constants in a single-molecule competition assay.

The ability of biophysicists to decipher the behavior of individual biomolecules has steadily improved over the past thirty years. However, it still remains unclear how an ensemble of data acquired at the single-molecule level compares with the data acquired on an ensemble of the same molecules. We here propose an assay to tackle this question in the context of dissociation equilibrium constant measurements. A sensor is built by engrafting a receptor and a ligand onto a flexible dsDNA scaffold and mounting this assembly on magnetic tweezers. This way, looking at the position of the magnetic bead enables one to determine in real-time if the two molecular partners are associated or not. Next, to quantify the affinity of the scrutinized single-receptor for a given competitor, various amounts of the latter molecule are introduced in solution and the equilibrium response of the sensor is monitored throughout the titration protocol. Proofs of concept are established for the binding of three rapamycin analogs to the FKBP12 cis-trans prolyl isomerase. For each of these drugs the mean affinity constant obtained on a ten of individual receptors agrees with the one previously determined in a bulk assay. Furthermore, experimental contingencies are sufficient to explain the dispersion observed over the single-molecule values.

Combining DNA scaffolds and acoustic force spectroscopy to characterize individual protein bonds.

Single-molecule data are of great significance in biology, chemistry, and medicine. However, new experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are still needed. Acoustic force spectroscopy is an emerging manipulation technique which generates acoustic waves to apply force in parallel on multiple microbeads tethered to a surface. We here exploit this configuration in combination with the recently developed modular junctured-DNA scaffold that has been designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measure its unbinding kinetics under force at the single-bond level. Special efforts are made in analyzing the data to identify potential pitfalls. We propose a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well-established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy to study the force-dependent rupture of a single-domain antibody with its antigen. Overall, we get a good agreement with the published parameters that have been obtained at zero force and population level. Thus, our technique offers single-molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.

Molecular scaffolds: when DNA becomes the hardware for single-molecule investigations.

Over the past few decades, single-molecule manipulation has been widely applied to the real-time analysis of biomolecular interactions. It has enabled researchers to decipher structure-function relationships for polymers, enzymes, and larger-scale molecular machines, in particular by harnessing force to probe both chemical and mechanical stabilities. Nucleic acids have played a central role in this effort because, in addition to their biological significance, they exhibit unique polymeric properties which have recast them as key components participating in numerous experimental designs. In this review, we introduce recent developments highlighting this dual nature of nucleic acids in biophysics, as objects of study but also as tools allowing novel approaches. More specifically, we present molecular scaffolds as an emerging concept and describe their use in single-molecule force spectroscopy. Aspects related to folding and noncovalent interactions will be presented in parallel to research in enzymology, with a focus on the acquisition of thermodynamic and kinetic data.

A modular DNA scaffold to study protein-protein interactions at single-molecule resolution.

The residence time of a drug on its target has been suggested as a more pertinent metric of therapeutic efficacy than the traditionally used affinity constant. Here, we introduce junctured-DNA tweezers as a generic platform that enables real-time observation, at the single-molecule level, of biomolecular interactions. This tool corresponds to a double-strand DNA scaffold that can be nanomanipulated and on which proteins of interest can be engrafted thanks to widely used genetic tagging strategies. Thus, junctured-DNA tweezers allow a straightforward and robust access to single-molecule force spectroscopy in drug discovery, and more generally in biophysics. Proof-of-principle experiments are provided for the rapamycin-mediated association between FKBP12 and FRB, a system relevant in both medicine and chemical biology. Individual interactions were monitored under a range of applied forces and temperatures, yielding after analysis the characteristic features of the energy profile along the dissociation landscape.

Understanding the mechanism of short-range electron transfer using an immobilized cupredoxin.

The hydrophobic patch of azurin (AZ) from Pseudomonas aeruginosa is an important recognition surface for electron transfer (ET) reactions. The influence of changing the size of this region, by mutating the C-terminal copper-binding loop, on the ET reactivity of AZ adsorbed on gold electrodes modified with alkanethiol self-assembled monolayers (SAMs) has been studied. The distance-dependence of ET kinetics measured by cyclic voltammetry using SAMs of variable chain length, demonstrates that the activation barrier for short-range ET is dominated by the dynamics of molecular rearrangements accompanying ET at the AZ-SAM interface. These include internal electric field-dependent low-amplitude protein motions and the reorganization of interfacial water molecules, but not protein reorientation. Interfacial molecular dynamics also control the kinetics of short-range ET for electrostatically and covalently immobilized cytochrome c. This mechanism therefore may be utilized for short-distance ET irrespective of the type of metal center, the surface electrostatic potential, and the nature of the protein-SAM interaction.

The influence of active site loop mutations on the thermal stability of azurin from Pseudomonas aeruginosa.

The copper site and overall structures of azurin (AZ) variants in which the amicyanin (AMI) and plastocyanin (PC) metal binding loops have been introduced, AZAMI and AZPC, respectively, are similar to that of AZ, whereas the loop conformations resemble those in the native proteins. To assess the influence of these loop mutations on stability, the thermal unfolding of AZAMI and AZPC has been investigated by differential scanning calorimetry, absorption and fluorescence spectroscopy. The calorimetric profiles of both variants exhibit a complex shape consisting of two endothermic peaks and an exothermic peak. The temperature of the maximum heat of absorption for the single endothermic peak is 82.7°C for AZ, whereas for AZAMI and AZPC the most intense endothermic peaks are at 74.9 and 68.1°C comparable to values for AMI and PC, respectively. Denaturation investigated using the temperature dependence of the absorbance at ∼600nm and Trp emission, also demonstrates decreased stability for both loop mutants. The thermal transition between the native and the denaturated states is irreversible, scan rate dependent and consistent with the two-state irreversible model. The structure of the active-site loop has a dramatic effect on the kinetic stability and the unfolding pathway of cupredoxins.

Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase.

Single-molecule measurements are a valuable tool for revealing details of enzyme mechanisms by enabling observation of unsynchronized behavior. However, this approach often requires immobilizing the enzyme on a substrate, a process which may alter enzyme behavior. We apply a microfluidic trapping device to allow, for the first time, prolonged solution-phase measurement of single enzymes in solution. Individual redox events are observed for single molecules of a blue nitrite reductase and are used to extract the microscopic kinetic parameters of the proposed catalytic cycle. Changes in parameters as a function of substrate concentration are consistent with a random sequential substrate binding mechanism.

Sonic Hedgehog-induced proliferation requires specific Gα inhibitory proteins.

Proliferation of cerebellar granular neuronal precursors (CGNPs) is mediated by Sonic Hedgehog (Shh), which activates the Patched and Smoothened (Smo) receptor complex. Although its protein sequence suggests that Smo is a G protein coupled receptor (GPCR), the evidence that this receptor utilizes heterotrimeric G proteins as downstream effectors is controversial. In Drosophila, Gα(i) is required for Hedgehog (Hh) activity, but the involvement of heterotrimeric G proteins in vertebrate Shh signaling has not yet been established. Here, we show that Shh-induced proliferation of rat CGNPs is enhanced strongly by the expression of the active forms of Gα(i/o) proteins (Gα(i1), Gα(i2), Gα(i3), and Gα(o)) but not by members of another class (Gα(12)) of heterotrimeric G proteins. Additionally, the mRNAs of these different Gα(i) members display specific expression patterns in the developing cerebellum; only Gα(i2) and Gα(i3) are substantially expressed in the outer external granular layer, where CGNPs proliferate. Consistent with this, Shh-induced proliferation of CGNPs is reduced significantly by knockdowns of Gα(i2) and Gα(i3) but not by silencing of other members of the Gα(i/o) class. Finally, our results demonstrate that Gα(i2) and Gα(i3) locate to the primary cilium when expressed in CGNP cultures. In summary, we conclude that the proliferative effects of Shh on CGNPs are mediated by the combined activity of Gα(i2) and Gα(i3) proteins.