Electrons, Photons, and Force: Quantitative Single-Molecule Measurements from Physics to Biology

Single-molecule measurement techniques have illuminated unprecedented details of chemical behavior, including observations of the motion of a single molecule on a surface, and even the vibration of a single bond within a molecule. Such measurements are critical to our understanding of entities ranging from single atoms to the most complex protein assemblies. We provide an overview of the strikingly diverse classes of measurements that can be used to quantify single-molecule properties, including those of single macromolecules and single molecular assemblies, and discuss the quantitative insights they provide. Examples are drawn from across the single-molecule literature, ranging from ultrahigh vacuum scanning tunneling microscopy studies of adsorbate diffusion on surfaces to fluorescence studies of protein conformational changes in solution

Single-molecule Förster resonance energy transfer study of protein dynamics under denaturing conditions

Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of the small enzyme ribonuclease HI (RNase H) in the presence of the chemical denaturant guanidinium chloride (GdmCl) using single-molecule fluorescence microscopy, with a particular focus on the characterization of the unfolded-state ensemble. A dye pair was specifically attached to the enzyme to measure structural changes through Förster resonance energy transfer (FRET). Enzyme immobilization on star-polymer surfaces that were specially developed for negligible interaction with folded and unfolded proteins enabled us to monitor conformational changes of individual proteins for several hundred seconds. FRET efficiency histograms were calculated from confocal scan images. They showed an expansion of the unfolded proteins with increasing GdmCl concentration. Cross-correlation analysis of donor and acceptor fluorescence intensity time traces from single molecules revealed reconfiguration of the polypeptide chain on a timescale of ≈20 μs at 1.7 M GdmCl. Slow conformational dynamics gave rise to characteristic, stepwise FRET efficiency changes. Transitions between folded and unfolded enzyme molecules occurred on the 100-s timescale, in excellent agreement with bulk denaturation experiments. Transitions between unfolded conformations were more frequent, with characteristic times of ≈2 s. These data were analyzed to obtain information on the free energy landscape of RNase H in the presence of chemical denaturants

Are Bidentate Ligands Really Better than Monodentate Ligands For Nanoparticles?

Coordinating ligands are widely used to vary the solubility and reactivity of nanoparticles for subsequent bioconjugation. Although long-term colloidal stability is enhanced by using bidentate coordinating ligands over monodentate ones, other properties such as nonspecific adsorption of target molecules and ligand exchange have not been quantified. In this study, we modified a near-infrared dye to serve as a highly sensitive reporter for nonspecific binding of thiolated target molecules to nanoparticle surfaces that are functionalized with monodentate or bidentate coordinated ligands. Specifically, we analyzed nonspecific binding mechanisms to quantum dots (QDs) by fitting the adsorption profiles to the Hill equation and the parameters are used to provide a microscopic picture of how ligand density and lability control nonspecific adsorption. Surprisingly, bidentate ligands are worse at inhibiting adsorption to QD surfaces at low target/QD ratios, although they become better as the ratio increases, but only if the nanoparticle surface area is large enough to overcome steric effects. This result highlights that a balance between ligand density and lability depends on the dentate nature of the ligands and controls how molecules in solution can coordinate to the nanoparticle surface. These results will have major implications for a range of applications in nanobiomedicine, bioconjugation, single molecule spectroscopy, self-assembly, and nano(photo)catalysis where both nonspecific and specific surface interactions play important roles. As an example, we tested the ability of monodentate and bidentate functionalized nanoparticles to resist nonspecific adsorption of IgG antibodies that contained free thiol groups at a 1:1 QD/IgG ratio and found that QDs with monodentate ligands did indeed result in lower nonspecific adsorption

Affinity of C-Reactive Protein toward FcγRI Is Strongly Enhanced by the γ-Chain

C-reactive protein (CRP), the prototype human acute phase protein, is widely regarded as a key player in cardiovascular disease, but the identity of its cellular receptor is still under debate. By using ultrasensitive confocal imaging analysis, we have studied CRP binding to transfected COS-7 cells expressing the high-affinity IgG receptor FcγRI. Here we show that CRP binds to FcγRI on intact cells, with a kd of 10 ± 3 μmol/L. Transfection of COS-7 cells with a plasmid coding for both FcγRI and its functional counterpart, the γ-chain, markedly increases CRP affinity to FcγRI, resulting in a kd of 0.35 ± 0.10 μmol/L. The affinity increase results from an ∼30-fold enhanced association rate coefficient. The pronounced enhancement of affinity by the γ-chain suggests its crucial involvement in the CRP receptor interaction, possibly by mediating interactions between the transmembrane moieties of the receptors. Dissociation of CRP from the cell surfaces cannot be detected throughout the time course of several hours and is thus extremely slow. Considering the pentameric structure of CRP, this result indicates that multivalent binding and receptor clustering are crucially involved in the interaction of CRP with nucleated cells