Electromechanical Photophysics of GFP Packed Inside Viral Protein Cages Probed by Force-Fluorescence Hybrid Single-Molecule Microscopy.

Packing biomolecules inside virus capsids has opened new avenues for the study of molecular function in confined environments. These systems not only mimic the highly crowded conditions in nature, but also allow their manipulation at the nanoscale for technological applications. Here, green fluorescent proteins are packed in virus-like particles derived from P22 bacteriophage procapsids. The authors explore individual virus cages to monitor their emission signal with total internal reflection fluorescence microscopy while simultaneously changing the microenvironment with the stylus of atomic force microscopy. The mechanical and electronic quenching can be decoupled by ≈10% each using insulator and conductive tips, respectively. While with conductive tips the fluorescence quenches and recovers regardless of the structural integrity of the capsid, with the insulator tips quenching only occurs if the green fluorescent proteins remain organized inside the capsid. The electronic quenching is associated with the coupling of the protein fluorescence emission with the tip surface plasmon resonance. In turn, the mechanical quenching is a consequence of the unfolding of the aggregated proteins during the mechanical disruption of the capsid.

Single-molecule imaging and manipulation of biomolecular machines and systems.

BACKGROUND: Biological molecular machines support various activities and behaviors of cells, such as energy production, signal transduction, growth, differentiation, and migration. SCOPE OF REVIEW: We provide an overview of single-molecule imaging methods involving both small and large probes used to monitor the dynamic motions of molecular machines in vitro (purified proteins) and in living cells, and single-molecule manipulation methods used to measure the forces, mechanical properties and responses of biomolecules. We also introduce several examples of single-molecule analysis, focusing primarily on motor proteins and signal transduction systems. MAJOR CONCLUSIONS: Single-molecule analysis is a powerful approach to unveil the operational mechanisms both of individual molecular machines and of systems consisting of many molecular machines. GENERAL SIGNIFICANCE: Quantitative, high-resolution single-molecule analyses of biomolecular systems at the various hierarchies of life will help to answer our fundamental question: "What is life?" This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato.

Pick-and-Place Assembly of Single Microtubules.

Intracellular transport is affected by the filament network in the densely packed cytoplasm. Biophysical studies focusing on intracellular transport based on microtubule-kinesin system frequently use in vitro motility assays, which are performed either on individual microtubules or on random (or simple) microtubule networks. Assembling intricate networks with high flexibility requires the manipulation of 25 nm diameter microtubules individually, which can be achieved through the use of pick-and-place assembly. Although widely used to assemble tiny objects, pick-and-place is not a common practice for the manipulation of biological materials. Using the high-level handling capabilities of microelectromechanical systems (MEMS) technology, tweezers are designed and fabricated to pick and place single microtubule filaments. Repeated picking and placing cycles provide a multilayered and multidirectional microtubule network even for different surface topographies. On-demand assembly of microtubules forms crossings at desired angles for biophysical studies as well as complex networks that can be used as nanotransport systems.

Convex lens-induced nanoscale templating.

We demonstrate a new platform, convex lens-induced nanoscale templating (CLINT), for dynamic manipulation and trapping of single DNA molecules. In the CLINT technique, the curved surface of a convex lens is used to deform a flexible coverslip above a substrate containing embedded nanotopography, creating a nanoscale gap that can be adjusted during an experiment to confine molecules within the embedded nanostructures. Critically, CLINT has the capability of transforming a macroscale flow cell into a nanofluidic device without the need for permanent direct bonding, thus simplifying sample loading, providing greater accessibility of the surface for functionalization, and enabling dynamic manipulation of confinement during device operation. Moreover, as DNA molecules present in the gap are driven into the embedded topography from above, CLINT eliminates the need for the high pressures or electric fields required to load DNA into direct-bonded nanofluidic devices. To demonstrate the versatility of CLINT, we confine DNA to nanogroove and nanopit structures, demonstrating DNA nanochannel-based stretching, denaturation mapping, and partitioning/trapping of single molecules in multiple embedded cavities. In particular, using ionic strengths that are in line with typical biological buffers, we have successfully extended DNA in sub-30-nm nanochannels, achieving high stretching (90%) that is in good agreement with Odijk deflection theory, and we have mapped genomic features using denaturation analysis.

Pasteur's Experiment Performed at the Nanoscale: Manual Separation of Chiral Molecules, One by One.

Understanding the principles of molecular recognition is a difficult task and calls for investigation of appropriate model systems. Using the manipulation capabilities of scanning tunneling microscopy (STM) we analyzed the chiral recognition in self-assembled dimers of helical hydrocarbons at the single molecule level. After manual separation of the two molecules of a dimer with a molecule-terminated STM tip on a Cu(111) surface, their handedness was subsequently determined with a metal atom-terminated tip. We find that these molecules strongly prefer to form heterochiral pairs. Our study shows that single molecule manipulation is a valuable tool to understand intermolecular recognition at surfaces.

Covalent Assembly and Characterization of Nonsymmetrical Single-Molecule Nodes.

The covalent linking of molecular building blocks on surfaces enables the construction of specific molecular nanostructures of well-defined shape. Molecular nodes linked to various entities play a key role in such networks, but represent a particular challenge because they require a well-defined arrangement of different building blocks. Herein, we describe the construction of a chemically and geometrically well defined covalent architecture made of one central node and three molecular wires arranged in a nonsymmetrical way and thus encoding different conjugation pathways. Very different architectures of either very limited or rather extended size were obtained depending on the building blocks used for the covalent linking process on the Au(111) surface. Electrical measurements were carried out by pulling individual molecular nodes with the tip of a scanning tunneling microscope. The results of this challenging procedure indicate subtle differences if the nodes are contacted at inequivalent termini.

Unravelling the Biophysical Properties of Chromatin Proteins and DNA Using Acoustic Force Spectroscopy.

Acoustic force spectroscopy (AFS) is a single-molecule micromanipulation technique that uses sound waves to exert force on surface-tethered DNA molecules in a microfluidic chamber. As large numbers of individual protein-DNA complexes are tracked in parallel, AFS provides insight into the individual properties of such complexes as well as their population averages. In this chapter, we describe in detail how to perform AFS experiments specifically on bare DNA, protein-DNA complexes, and how to extract their (effective) persistence length and contour length from force-extension relations.

High-resolution optical tweezers for single-molecule manipulation.

Forces hold everything together and determine its structure and dynamics. In particular, tiny forces of 1-100 piconewtons govern the structures and dynamics of biomacromolecules. These forces enable folding, assembly, conformational fluctuations, or directional movements of biomacromolecules over sub-nanometer to micron distances. Optical tweezers have become a revolutionary tool to probe the forces, structures, and dynamics associated with biomacromolecules at a single-molecule level with unprecedented resolution. In this review, we introduce the basic principles of optical tweezers and their latest applications in studies of protein folding and molecular motors. We describe the folding dynamics of two strong coiled coil proteins, the GCN4-derived protein pIL and the SNARE complex. Both complexes show multiple folding intermediates and pathways. ATP-dependent chromatin remodeling complexes translocate DNA to remodel chromatin structures. The detailed DNA translocation properties of such molecular motors have recently been characterized by optical tweezers, which are reviewed here. Finally, several future developments and applications of optical tweezers are discussed. These past and future applications demonstrate the unique advantages of high-resolution optical tweezers in quantitatively characterizing complex multi-scale dynamics of biomacromolecules.

Transverse Magnetic Tweezers Allowing Coincident Epi-Fluorescence Microscopy on Horizontally Extended DNA.

Longitudinal magnetic tweezers (L-MT) have seen wide-scale adoption as the tool of choice for stretching and twisting a single DNA molecule. They are also used to probe topological changes in DNA as a result of protein binding and enzymatic activity. However, in the longitudinal configuration, the DNA molecule is extended perpendicular to the imaging plane. As a result, it is only possible to infer biological activity from the motion of the tethered paramagnetic microsphere. Described here is a "transverse" magnetic tweezers (T-MT) geometry featuring simultaneous control of DNA extension and spatially coincident video-rate epi-fluorescence imaging. Unlike in L-MT, DNA tethers in T-MT are extended parallel to the imaging plane between two micron-sized spheres, and importantly protein targets on the DNA can be localized using fluorescent nanoparticles. The T-MT can manipulate a long DNA construct at molecular extensions approaching the contour length defined by B-DNA helical geometry, and the measured entropic elasticity agrees with the wormlike chain model (force <35 pN). By incorporating a torsionally constrained DNA tether, the T-MT would allow both the relative extension and twist of the tether to be manipulated, while viewing far-red emitting fluorophore-labeled targets. This T-MT design has the potential to enable the study of DNA binding and remodeling processes under conditions of constant force and defined torsional stress.

DNA-binding proteins studied by mechanical manipulation and AFM imaging of single DNA molecules.

The functions of DNA-binding proteins are dependent on protein-induced DNA distortion, the binding preference to special sequences, DNA secondary structures, the binding kinetics and the binding affinity. Recent rapid progress in single-molecule imaging and mechanical manipulation technologies have made it possible to directly probe the DNA binding by proteins, footprint the positions of the bound proteins on DNA, quantify the kinetics and the affinity of protein-DNA interactions, and study the interplay of protein binding with DNA conformation and DNA topology. Here, we review the applications of an integrated approach where the single-DNA imaging using atomic force microscopy and the mechanical manipulation of single DNA molecules are combined to study the DNA-protein interactions. We also provide our views on how these findings yield new insights into understanding the roles of several essential DNA architectural proteins.

Using Magnetic Tweezers to Unravel the Mechanism of the G-quadruplex Binding and Unwinding Activities of DHX36 Helicase.

Single-molecule manipulation methods are useful techniques to probe the interactions of proteins and nucleic acid structures. Here, we describe the magnetic tweezers-based single-molecule investigation of the binding of helicases to G-quadruplex structures and their ATP-dependent unwinding activity, using DHX36 (also known as RHAU and G4R1) helicase and a DNA G-quadruplex structure for an example. We specifically emphasize on the principle and method to probe the interactions between DHX36 and the DNA G-quadruplex in different intermediate states during an ATPase cycle of DHX36, based on detecting the DHX36-induced changes in the lifetime of the DNA G-quadruplex under tension. The principle of the measurement can be broadly extended to the studies of other DNA or RNA G-quadruplex helicases.

Optical Tweezers to Investigate the Structure and Energetics of Single-Stranded DNA-Binding Protein-DNA Complexes.

Optical tweezers enable the isolation and mechanical manipulation of individual nucleoprotein complexes. Here, we describe how to use this technique to interrogate the mechanical properties of individual protein-DNA complexes and extract information about their overall structural organization.

Measurements of Real-Time Replication Kinetics of DNA Polymerases on ssDNA Templates Coated with Single-Stranded DNA-Binding Proteins.

Optical tweezers can monitor and control the activity of individual DNA polymerase molecules in real time, providing in this way unprecedented insight into the complex dynamics and mechanochemical processes that govern their operation. Here, we describe an optical tweezers-based assay to determine at the single-molecule level the effect of single-stranded DNA-binding proteins (SSB) on the real-time replication kinetics of the human mitochondrial DNA polymerase during the synthesis of the lagging strand.

Investigation of edge states in artificial graphene nano-flakes.

Graphene nano-flakes (GNFs) are predicted to host spin-polarized metallic edge states, which are envisioned for exploration of spintronics at the nanometer scale. To date, experimental realization of GNFs is only in its infancy because of the limitation of precise cutting or synthesizing methods at the nanometer scale. Here, we use low temperature scanning tunneling microscope to manipulate coronene molecules on a Cu(111) surface to build artificial triangular and hexagonal GNFs with either zigzag or armchair type of edges. We observe that an electronic state at the Dirac point emerges only in the GNFs with zigzag edges and localizes at the outmost lattice sites. The experimental results agree well with the tight-binding calculations. Our work renders an experimental confirmation of the predicated edge states of the GNFs.

Studying heat shock proteins through single-molecule mechanical manipulation.

Imbalances of cellular proteostasis are linked to ageing and human diseases, including neurodegenerative and neuromuscular diseases. Heat shock proteins (HSPs) and small heat shock proteins (sHSPs) together form a crucial core of the molecular chaperone family that plays a vital role in maintaining cellular proteostasis by shielding client proteins against aggregation and misfolding. sHSPs are thought to act as the first line of defence against protein unfolding/misfolding and have been suggested to act as "sponges" that rapidly sequester these aberrant species for further processing, refolding, or degradation, with the assistance of the HSP70 chaperone system. Understanding how these chaperones work at the molecular level will offer unprecedented insights for their manipulation as therapeutic avenues for the treatment of ageing and human disease. The evolution in single-molecule force spectroscopy techniques, such as optical tweezers (OT) and atomic force microscopy (AFM), over the last few decades have made it possible to explore at the single-molecule level the structural dynamics of HSPs and sHSPs and to examine the key molecular mechanisms underlying their chaperone activities. In this paper, we describe the working principles of OT and AFM and the experimental strategies used to employ these techniques to study molecular chaperones. We then describe the results of some of the most relevant single-molecule manipulation studies on HSPs and sHSPs and discuss how these findings suggest a more complex physiological role for these chaperones than previously assumed.

Experimental and theoretical energetics of walking molecular motors under fluctuating environments.

Molecular motors are nonequilibrium open systems that convert chemical energy to mechanical work. Their energetics are essential for various dynamic processes in cells, but largely remain unknown because fluctuations typically arising in small systems prevent investigation of the nonequilibrium behavior of the motors in terms of thermodynamics. Recently, Harada and Sasa proposed a novel equality to measure the dissipation of nonequilibrium small systems. By utilizing this equality, we have investigated the nonequilibrium energetics of the single-molecule walking motor kinesin-1. The dissipation from kinesin movement was measured through the motion of an attached probe particle and its response to external forces, indicating that large hidden dissipation exists. In this short review, aiming to readers who are not familiar with nonequilibrium physics, we briefly introduce the theoretical basis of the dissipation measurement as well as our recent experimental results and mathematical model analysis and discuss the physiological implications of the hidden dissipation in kinesin. In addition, further perspectives on the efficiency of motors are added by considering their actual working environment: living cells.

Single-Molecule Optical Tweezers Study of Regulated SNARE Assembly.

Intracellular membrane fusion mediates material and information exchange among different cells or cellular compartments with high accuracy and spatiotemporal resolution. Fusion is driven by ordered folding and assembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) and regulated by many other proteins. Understanding regulated SNARE assembly is key to dissecting mechanisms and physiologies of various fusion processes and their associated diseases. Yet, it remains challenging to study regulated SNARE assembly using traditional ensemble-based experimental approaches. Here, we describe our new method to measure the energy and kinetics of neuronal SNARE assembly in the presence of α-SNAP, using a single-molecule manipulation approach based on high-resolution optical tweezers. Detailed experimental protocols and methods of data analysis are shown. This approach can be widely applied to elucidate the effects of regulatory proteins on SNARE assembly and membrane fusion.

Measuring Unzipping and Rezipping of Single Long DNA Molecules with Optical Tweezers.

The unwinding of double-stranded DNA is a frequently occurring event during the cellular processes of DNA replication, repair, and transcription. To help further investigate properties of this fundamental process as well as to study proteins acting on unzipped DNA at the single molecule level, we describe a novel method for efficient preparation of long DNA constructs (arbitrary sequences of many kilobasepairs (kbp) in length) that can be forcibly unzipped and manipulated with optical tweezers or other single-molecule manipulation techniques. This method utilizes PCR, a nicking endonuclease, and strand displacement synthesis by the Klenow fragment of DNA polymerase I to introduce labeled nucleotides at appropriate positions to facilitate unzipping of the DNA by application of force. We also describe various optical tweezers measurement modes for measuring DNA unzipping and rezipping. These methods have applications to studying helicases and DNA binding proteins.

Single-Molecule Measurements of Motor-Driven Viral DNA Packaging in Bacteriophages Phi29, Lambda, and T4 with Optical Tweezers.

Viral DNA packaging is a required step in the assembly of many dsDNA viruses. A molecular motor fueled by ATP hydrolysis packages the viral genome to near crystalline density inside a preformed prohead shell in ~5 min at room temperature. We describe procedures for measuring the packaging of single DNA molecules into single viral proheads with optical tweezers. Three viral packaging systems are described in detail: bacteriophages phi29 (φ29), lambda (λ), and T4. Two different approaches are described: (1) With φ29 and T4, prohead-motor complexes can be preassembled in bulk and packaging can be initiated in the optical tweezers by "feeding" a single DNA molecule to one of the complexes; (2) With φ29 and λ, packaging can be initiated in bulk then stalled, and a single prohead-motor-DNA complex can then be captured with optical tweezers and restarted. In both cases, the prohead is ultimately attached to one trapped microsphere and the end of the DNA being packaged is attached to a second trapped microsphere such that packaging of the DNA pulls the two microspheres together and the rate of packaging and force generated by the motor is directly measured in real time. These protocols allow for the effect of many experimental parameters on packaging dynamics to be studied such as temperature, ATP concentration, ionic conditions, structural changes to the DNA substrate, and mutations in the motor proteins. Procedures for capturing microspheres with the optical traps and different measurement modes are also described.

Unraveling the Biophysical Properties of Chromatin Proteins and DNA Using Acoustic Force Spectroscopy.

Acoustic Force Spectroscopy (AFS) is a single-molecule micromanipulation technique that uses sound waves to exert force on surface-tethered DNA molecules in a microfluidic chamber. As large numbers of individual protein-DNA complexes are tracked in parallel, AFS provides insight into the individual properties of such complexes as well as their population averages. In this chapter, we describe in detail how to perform AFS experiments specifically on bare DNA, protein-DNA complexes, and how to extract their (effective) persistence length and contour length from force-extension relations.