Identification of sugar isomers by single-molecule force spectroscopy

The nanomechanical properties of beta-galactan, a 1 --> 4 linked beta-d-galactose polysaccharide, were investigated with AFM-based single-molecule force spectroscopy. AFM captured a unique plateau at 640 pN in the force spectrogram of beta-galactan, which is significantly different than the plateau at 280 pN in the force spectrogram of amylose. Thus, our results demonstrate that force spectroscopy is able to discriminate between sugar isomers in which axial and equatorial bonds at C1 and C4 are swapped.     

Interaction between dendrons directly studied by single-molecule force spectroscopy

In this article, we have investigated the interaction between two poly(benzyl ether) dendrons directly by single-molecule force spectroscopy. For this purpose, one dendron was immobilized on an AFM tip through a poly(ethylene glycol) (PEG) spacer, and the other dendron was anchored on a gold substrate as a self-assembled monolayer. Two dendrons approached and then interacted with each other when the AFM tip and the substrate moved close together. The rupture force between dendrons was measured while the AFM tip and the substrate separated. PEG as a flexible spacer can function as a length window for recognizing the force signals and avoiding the disturbance of the interaction between the AFM tip and the substrate. The interaction between two first-generation dendrons is measured to be about 224 pN at a force loading rate of 40 nN/s. The interaction between second- and first-generation dendrons rises to 315 pN at the same loading rate. Such interactions depend on the force loading rate in the range of several to hundreds of nanonewtons per second, indicating that the rupture between dendrons is a dynamic process. The study of the interaction between surface-bound dendrons of different generations provides a model system for understanding the surface adhesion of molecules with multiple branches. In addition, this multiple-branch molecule may be used to mimic the sticky feet of geckos as a man-made adhesive.     

Influence of pulling handles and device stiffness in single-molecule force spectroscopy

In single-molecule force spectroscopy, individual molecules and complexes are often stretched by pulling devices via intervening molecular handles. Accurate interpretation of measurements from such experiments in terms of the underlying energy landscape, defined by activation barriers and intrinsic rates of transition, relies on our understanding, and proper theoretical treatment, of the effects of the pulling device and handle. Here, we present a framework based on Kramers' theory that elucidates the dependence of measured rupture forces and rates on the pulling device stiffness and attributes of the handle, contour length and persistence length. We also introduce a simple analytic model that improves prediction of activation barriers and intrinsic rates for all device stiffnesses and handle properties, thus allowing for a more reliable interpretation of experiments. Our analyses also suggests intuitive ways of displaying the measured force spectra for proper prognosis of device and handle effects and provides the range of device and handle attributes over which these effects can be neglected.     

Facile method of constructing polyproteins for single-molecule force spectroscopy studies

Constructing polyproteins consisting of identical tandem repeats of proteins provides an unambiguous method of investigating the mechanical properties of proteins at the single-molecule level using force spectroscopy techniques. Here we report a maleimide-thiol coupling-based facile method of constructing polyproteins for single-molecule force spectroscopy studies on the mechanical properties of proteins. This method allows for the construction of polyproteins in an efficient fashion under room temperature. The resultant thioether bonds are resistant to reduction and make it possible to carry out single-molecule force spectroscopy studies under various redox conditions. This novel method complements existing polyprotein engineering methods and can be easily applied to a wide variety of proteins.     

Mori-Zwanzig memory analysis in single-molecule spectroscopy

We study the dynamical behavior of the Rhodospirillum molischianum LH2 complex based on intensity time series obtained from single-molecule spectroscopy experiments. This is achieved by reconstructing the memory function describing the time-dependent fluctuations of the excited states. We conclude that the apparent stochastic evolution of the dynamics is controlled by at least two different non-Markovian main processes.     

Correction of systematic errors in single-molecule force spectroscopy with polymeric tethers by atomic force microscopy

Single-molecule force spectroscopy has become a valuable tool for the investigation of intermolecular energy landscapes for a wide range of molecular associations. Atomic force microscopy (AFM) is often used as an experimental technique in these measurements, and the Bell-Evans model is commonly used in the statistical analysis of rupture forces. Most applications of the Bell-Evans model consider a constant loading rate of force applied to the intermolecular bond. The data analysis is often inconsistent because either the probe velocity or the apparent loading rate is being used as an independent parameter. These approaches provide different results when used in AFM-based experiments. Significant variations in results arise from the relative stiffness of the AFM force sensor in comparison with the stiffness of polymeric tethers that link the molecules under study to the solid surfaces. An analytical model presented here accounts for the systematic errors in force-spectroscopy parameters arising from the nonlinear loading induced by polymer tethers. The presented analytical model is based on the Bell-Evans model of the kinetics of forced dissociation and on the asymptotic models of tether stretching. The two most common data reduction procedures are analyzed, and analytical expressions for the systematic errors are provided. The model shows that the barrier width is underestimated and that the dissociation rate is significantly overestimated when force-spectroscopy data are analyzed without taking into account the elasticity of the polymeric tether. Systematic error estimates for asymptotic freely jointed chain and wormlike chain polymer models are given for comparison. The analytical model based on the asymptotic freely jointed chain stretching is employed to analyze and correct the results of the double-tether force-spectroscopy experiments of disjoining "hydrophobic bonds" between individual hexadecane molecules that are covalently tethered via poly(ethylene glycol) linkers of different lengths to the substrates and to the AFM probes. Application of the correction algorithm decreases the spread of the data from the mean value, which is particularly important for measurements of the dissociation rate, and increases the barrier width to 0.43 nm, which might be indicative of the theoretically predicted hydrophobic dewetting.     

Reverse engineering of an affinity-switchable molecular interaction characterized by atomic force microscopy single-molecule force spectroscopy

Tunable and switchable interaction between molecules is a key for regulation and control of cellular processes. The translation of the underlying physicochemical principles to synthetic and switchable functional entities and molecules that can mimic the corresponding molecular functions is called reverse molecular engineering. We quantitatively investigated autoinducer-regulated DNA-protein interaction in bacterial gene regulation processes with single atomic force microscopy (AFM) molecule force spectroscopy in vitro, and developed an artificial bistable molecular host-guest system that can be controlled and regulated by external signals (UV light exposure and thermal energy). The intermolecular binding functionality (affinity) and its reproducible and reversible switching has been proven by AFM force spectroscopy at the single-molecule level. This affinity-tunable optomechanical switch will allow novel applications with respect to molecular manipulation, nanoscale rewritable molecular memories, and/or artificial ion channels, which will serve for the controlled transport and release of ions and neutral compounds in the future.     

Time and distance dependence of reversible polymer bridging followed by single-molecule force spectroscopy

Polymer bridging between surfaces plays an important role in a range of fundamental processes in the material and life sciences. Bridges formed by main-chain reversible polymers differ from their covalent analogs in that they can dynamically adjust their size and shape in response to external stimuli and have the potential to reform following bond scission. In this work, the time and distance dependence of main-chain reversible polymer bridge formation are studied using an atomic force microscope. The bridging process was studied using single-molecule force spectroscopy, and its dependence on the distance between surfaces and equilibration time was probed. The number of bridges formed decreases as the gap width increases, from approximately 2 bridges per 14 s equilibration at separations of 5-15 nm to approximately 0.5 bridges per 14 s equilibration at separations of 35-45 nm. The kinetics of bridge formation appear to be slightly faster at smaller separations.     

Quantitative microfluidic biomolecular analysis for systems biology and medicine

In the postgenome era, biology and medicine are rapidly evolving towards quantitative and systems studies of complex biological systems. Emerging breakthroughs in microfluidic technologies and innovative applications are transforming systems biology by offering new capabilities to address the challenges in many areas, such as single-cell genomics, gene regulation networks, and pathology. In this review, we focus on recent progress in microfluidic technology from the perspective of its applications to promoting quantitative and systems biomolecular analysis in biology and medicine.     

TOTO binding affinity analysis using single-molecule fluorescence spectroscopy

The sequence dependence of the double-stranded DNA (dsDNA)-binding affinity of TOTO, a thiazole orange dimer that functions as a DNA-intercalating fluorophore, was measured using single-molecule methods. An analysis was performed of the distribution of excited-state lifetimes of single molecules of TOTO intercalated into dsDNA fragments containing four-base pair sequences shown previously to have high affinity for TOTO under conditions used in nuclear magnetic resonance (NMR) spectroscopy. For the current studies, the putative binding sites were located centrally in 30-base pair-long dsDNA fragments in which the remaining sequence was either all poly-AT or poly-GC. The lifetime of TOTO fluorescence when bound to these fragments was entirely determined by the background sequence, i.e. DNA fragments with a poly-AT background predominantly gave a fluorescence lifetime of 1.7 ns, whereas DNA fragments with a poly-GC background gave a lifetime of 2.0 ns, independent of the presence or absence of the putative binding sequence. By performing competitive binding experiments in which these DNA fragments competed for TOTO binding with pure poly-AT fragments and using single-molecule fluorescence methods to determine the number of each type of DNA fragment having a TOTO bound in an equilibrium mixture, the relative binding affinity of each putative binding site was determined. The results of these experiments showed clearly that TOTO has no preference for binding to the putative binding sites over binding poly-AT or poly-GC under the conditions of these measurements. This suggests that there is very little sequence dependence of TOTO binding under conditions that would likely predominate in most biological applications of this intercalating dye.     

Energy landscapes of biomolecular adhesion and receptor anchoring at interfaces explored with dynamic force spectroscopy

Beyond covalent connections within protein and lipid molecules, weak noncovalent interactions between large molecules govern properties of cellular structure and interfacial adhesion in biology. These bonds and structures have limited lifetimes and so will fail under any level of force if pulled on for the right length of time. As such, the strength of interaction is the level of force most likely to disrupt a bond on a particular time scale. For instance, strength is zero on time scales longer than the natural lifetime for spontaneous dissociation. On the other hand, if driven to unbind or change structure on time scales shorter than needed for diffusive relaxation, strength will reach an adiabatic limit set by the maximum gradient in a potential of mean force. Over the enormous span of time scales between spontaneous dissociation and adiabatic detachment, theory predicts that bond breakage under steadily rising force occurs most frequently at a force determined by the rate of loading. Moreover, the continuous plot (spectrum) of strength expressed on a scale of loge(loading rate) provides a map of the prominent barriers traversed in the energy landscape along the force-driven pathway and reveals the differences in energy between barriers. Illustrated with results from recent laboratory measurements, dynamic strength spectra provide a new view into the inner complexity of receptor-ligand interactions and receptor lipid anchoring.     

Mechanically tightening,untying and retying a protein trefoil knot by single-molecule force spectroscopy

Knotted conformation is one of the most surprising topological features found in proteins, and understanding the folding mechanism of such knotted proteins remains a challenge. Here, we used optical tweezers (OT) to investigate the mechanical unfolding and folding behavior of a knotted protein Escherichia coli tRNA (guanosine-1) methyltransferase (TrmD). We found that when stretched from its N- and C-termini, TrmD can be mechanically unfolded and stretched into a tightened trefoil knot, which is composed of ca. 17 residues. Stretching of the unfolded TrmD involved a compaction process of the trefoil knot at low forces. The unfolding pathways of the TrmD were bifurcated, involving two-state and three-state pathways. Upon relaxation, the tightened trefoil knot loosened up first, leading to the expansion of the knot, and the unfolded TrmD can then fold back to its native state efficiently. By using an engineered truncation TrmD variant, we stretched TrmD along a pulling direction to allow us to mechanically unfold TrmD and untie the trefoil knot. We found that the folding of TrmD from its unfolded polypeptide without the knot is significantly slower. The knotting is the rate-limiting step of the folding of TrmD. Our results highlighted the critical importance of the knot conformation for the folding and stability of TrmD, offering a new perspective to understand the role of the trefoil knot in the biological function of TrmD.

Optical tweezers are used to stretch a knotted protein along different directions to probe its unfolding–folding behaviors, and the conformational change of its knot structure.      

Investigating the specific interactions between carbonic anhydrase and a sulfonamide inhibitor by single-molecule force spectroscopy

In this communication, we report on the interaction landscape of an active site-specific enzyme-inhibitor complex by single-molecule force spectroscopy. Electrostatic immobilization was employed to orient a carbonic anhydrase enzyme on a positively charged surface so its active site is pointing upward. This approach to immobilization effectively increases the number of specific interactions measured between the zinc ion of the active site on carbonic anhydrase and a sulfonamide inhibitor tethered to an atomic force microscope (AFM) probe. Further, it reduces the time required for data collection and thereby minimizes the possible mechanical damage to the probe and contamination of the enzyme surface. The rupture force measured at various loading rates is interpreted in terms of a single energy barrier for the carbonic anhydrase enzyme-sulfonamide inhibitor complex from which the kinetic and thermodynamic parameters were estimated on the basis of microscopic models and were compared to the Bell-Evans model. The dissociation rate for the enzyme-inhibitor complex was found to be significantly faster (~35 times) than the natural spontaneous dissociation rate.     

EMSA and single-molecule force spectroscopy study of interactions between Bacillus subtilis single-stranded DNA-binding protein and single-stranded DNA

In this article, interactions between Bacillus subtilis single-stranded DNA binding proteins (BsSSB) and single-stranded DNA (ssDNA) were systematically studied. The effect of different molar ratios between BsSSB and ssDNA on their binding modes was first investigated by electrophoretic mobility shift assays (EMSAs). It is found that a high molar ratio of BsSSB to ssDNA can produce BsSSB-ssDNA complexes formed in the mode of two proteins binding one 65-nt (nucleotide) ssDNA whereas a low molar ratio facilitates the formation of BsSSB-ssDNA complexes in the mode of one protein binding one 65-nt ssDNA. Furthermore, two binding modes are in dynamic equilibrium. The unbinding force of BsSSB-ssDNA complexes was measured quantitatively in solutions with different salt concentrations by using AFM-based single-molecule force spectroscopy (SMFS). Our results show that the unbinding force is about 10 pN higher at high salt concentration (0.5 M NaCl) than at low salt concentration (0.1 M NaCl) and the lifetime of BsSSB-ssDNA complexes at high salt concentration is twice as long as that at low salt concentration. These results indicate that more tightly packed BsSSB-ssDNA complexes can form at high salt (0.5 M NaCl) concentration. In addition, the results of EMSA show that ssDNA, which is bound to BsSSB, can dissociate from BsSSB in the presence of the cDNA strand, indicating the dynamic nature of BsSSB-ssDNA interactions.     

Multiphoton excited fluorescence spectroscopy of biomolecular systems

Recent work on the emerging application of multiphoton excitation to fluorescence studies of biomolecular dynamics and structure is reviewed. The fundamental principles and experimental techniques of multiphoton excitation are outlined, fluorescence lifetimes, anisotropy and spectra in membranes, proteins, hydrocarbons, skin, tissue and metabolites are featured, and future opportunities are highlighted.     

Rupture force analysis and the associated systematic errors in force spectroscopy by AFM

Force spectroscopy is a new and valuable tool in physical chemistry and biophysics. However, data analysis has yet to be standardized, hindering the advancement of the technique. In this article, treatment of the rupture forces is described in the framework of the Bell-Evans model, and the systematic errors associated with the tether effect for approaches that utilize the most probable, the median, and the mean rupture forces are compared. It is shown that significant systematic errors in the dissociation rate can result from nonlinear loading with polymeric tethers even if the apparent loading rate is used in the analysis. Analytical expressions for the systematic errors are provided for the most probable and median forces. The use of these expressions to correct the associated systematic errors is illustrated by the analysis of the measured rupture forces between single hexadecane molecules in water. It is noted that the measured distributions of rupture forces often contain high forces that are unaccounted for by theoretical models. Experimental data indicate that the most significant effect of the high forces "tail" is on the dissociation rate obtained from the median force analysis whereas the barrier width appears to be unaffected.