Using Single-Molecule Approaches to Understand the Molecular Mechanisms of Heat-Shock Protein Chaperone Function

The heat-shock proteins (Hsp) are a family of molecular chaperones, which collectively form a network that is critical for the maintenance of protein homeostasis. Traditional ensemble-based measurements have provided a wealth of knowledge on the function of individual Hsps and the Hsp network; however, such techniques are limited in their ability to resolve the heterogeneous, dynamic and transient interactions that molecular chaperones make with their client proteins. Single-molecule techniques have emerged as a powerful tool to study dynamic biological systems, as they enable rare and transient populations to be identified that would usually be masked in ensemble measurements. Thus, single-molecule techniques are particularly amenable for the study of Hsps and have begun to be used to reveal novel mechanistic details of their function. In this review, we discuss the current understanding of the chaperone action of Hsps and how gaps in the field can be addressed using single-molecule methods. Specifically, this review focuses on the ATP-independent small Hsps and the broader Hsp network and describes how these dynamic systems are amenable to single-molecule techniques.     

Nano-Precision Tweezers for Mechanosensitive Proteins and Beyond

Mechanical forces play pivotal roles in regulating cell shape, function, and fate. Key players that govern the mechanobiological interplay are the mechanosensitive proteins found on cell membranes and in cytoskeleton. Their unique nanomechanics can be interrogated using single-molecule tweezers, which can apply controlled forces to the proteins and simultaneously measure the ensuing structural changes. Breakthroughs in high-resolution tweezers have enabled the routine monitoring of nanometer-scale, millisecond dynamics as a function of force. Undoubtedly, the advancement of structural biology will be further fueled by integrating static atomic-resolution structures and their dynamic changes and interactions observed with the force application techniques. In this minireview, we will introduce the general principles of single-molecule tweezers and their recent applications to the studies of force-bearing proteins, including the synaptic proteins that need to be categorized as mechanosensitive in a broad sense. We anticipate that the impact of nano-precision approaches in mechanobiology research will continue to grow in the future.     

Protein nanomechanics in biological context

How proteins respond to pulling forces, or protein nanomechanics, is a key contributor to the form and function of biological systems. Indeed, the conventional view that proteins are able to diffuse in solution does not apply to the many polypeptides that are anchored to rigid supramolecular structures. These tethered proteins typically have important mechanical roles that enable cells to generate, sense, and transduce mechanical forces. To fully comprehend the interplay between mechanical forces and biology, we must understand how protein nanomechanics emerge in living matter. This endeavor is definitely challenging and only recently has it started to appear tractable. Here, I introduce the main in vitro single-molecule biophysics methods that have been instrumental to investigate protein nanomechanics over the last 2 decades. Then, I present the contemporary view on how mechanical force shapes the free energy of tethered proteins, as well as the effect of biological factors such as post-translational modifications and mutations. To illustrate the contribution of protein nanomechanics to biological function, I review current knowledge on the mechanobiology of selected muscle and cell adhesion proteins including titin, talin, and bacterial pilins. Finally, I discuss emerging methods to modulate protein nanomechanics in living matter, for instance by inducing specific mechanical loss-of-function (mLOF). By interrogating biological systems in a causative manner, these new tools can contribute to further place protein nanomechanics in a biological context.     

Single-molecule approaches to prion protein misfolding

The structural conversion of the prion protein PrP into a transmissible, misfolded form is the central element of prion disease, yet there is little consensus as to how it occurs. Key aspects of conversion into the diseased state remain unsettled, from details about the earliest stages of misfolding such as the involvement of partially- or fully-unfolded intermediates to the structure of the infectious state. Part of the difficulty in understanding the structural conversion arises from the complexity of the underlying energy landscapes. Single molecule methods provide a powerful tool for probing complex folding pathways as in prion misfolding, because they allow rare and transient events to be observed directly. We discuss recent work applying single-molecule probes to study misfolding in prion proteins, and what it has revealed about the folding dynamics of PrP that may underlie its unique behavior. We also discuss single-molecule studies probing the interactions that stabilize non-native structures within aggregates, pointing the way to future work that may help identify the microscopic events triggering pathogenic conversion. Although single-molecule approaches to misfolding are relatively young, they have a promising future in prion science.     

NMR spectroscopy of G-quadruplexes

G-rich DNA and RNA sequences can form four-stranded structures called G-quadruplexes. Such structures have gained significant interest in the past decade with increasing evidence of their biological role. G-quadruplex structures can be polymorphic and dynamic. NMR spectroscopy has played an important role in G-quadruplex research. Here we review on the application of NMR techniques to study structure, dynamics and interaction of G-quadruplexes.     

An Insight into Biomolecular Flexibility: Its Measuring,Modeling and Regulating on Function at Single Molecule Level

The protein structure-function paradigm implies that the structure of a protein defines its function. Crystallization techniques such as X-ray, electron microscopy (EM) and nuclear magnetic resonance (NMR) have been applied to resolve the crystal structure of numerous proteins, provided beautiful and informative models of proteins. However, proteins are not intrinsically in static state but in dynamic state, which is lack in crystal models. The protein flexibility, a key mechanical property of proteins, plays important roles in various biological processes, such as ligand-receptor interaction, signaling transduction, substrate recognition and post-translational modifications. Advanced time-resolved crystallography has been developed recent years to visualize and characterize the dynamic of proteins and reviewed in literatures. In the present review, we will focus on the single-molecule based techniques and theoretical methods in determining the flexibility of proteins, exhibit some interest examples of proteins and DNA molecular flexibility to their functions, and provide an insight in molecular flexibility from the biomechanics point of view.     

Configurational entropy modulates the mechanical stability of protein GB1

Configurational entropy plays important roles in defining the thermodynamic stability as well as the folding/unfolding kinetics of proteins. Here we combine single-molecule atomic force microscopy and protein engineering techniques to directly examine the role of configurational entropy in the mechanical unfolding kinetics and mechanical stability of proteins. We used a small protein, GB1, as a model system and constructed four mutants that elongate loop 2 of GB1 by 2, 5, 24 and 46 flexible residues, respectively. These loop elongation mutants fold properly as determined by far-UV circular dichroism spectroscopy, suggesting that loop 2 is well tolerant of loop insertions without affecting GB1′s native structure. Our single-molecule atomic force microscopy results reveal that loop elongation decreases the mechanical stability of GB1 and accelerates the mechanical unfolding kinetics. These results can be explained by the loss of configurational entropy upon closing an unstructured flexible loop using classical polymer theory, highlighting the important role of loop regions in the mechanical unfolding of proteins. This study not only demonstrates a general approach to investigating the structural deformation of the loop regions in mechanical unfolding transition state, but also provides the foundation to use configurational entropy as an effective means to modulate the mechanical stability of proteins, which is of critical importance towards engineering artificial elastomeric proteins with tailored nanomechanical properties.     

A conserved strategy for structure change and energy transduction in Hsp104 and other AAA+ protein motors

The superfamily of massively large AAA+ protein molecular machines functions to convert the chemical energy of cytosolic ATP into physicomechanical form and use it to perform an extraordinary number of physical operations on proteins, nucleic acids, and membrane systems. Cryo-EM studies now reveal some aspects of substrate handling at high resolution, but the broader interpretation of AAA+ functional properties is still opaque. This paper integrates recent hydrogen exchange results for the typical AAA+ protein Hsp104 with prior information on several near and distantly related others. The analysis points to a widely conserved functional strategy. Hsp104 cycles through a long-lived loosely-structured energy-input “open” state that releases spent ADP and rebinds cytosolic ATP. ATP-binding energy is transduced by allosteric structure change to poise the protein at a high energy level in a more tightly structured “closed” state. The briefly occupied energy-output closed state binds substrate strongly and is catalytically active. ATP hydrolysis permits energetically downhill structural relaxation, which is coupled to drive energy-requiring substrate processing. Other AAA+ proteins appear to cycle through states that are analogous functionally if not in structural detail. These results revise the current model for AAA+ function, explain the structural basis of single-molecule optical tweezer kinetic phases, identify the separate energetic roles of ATP binding and hydrolysis, and specify a sequence of structural and energetic events that carry AAA+ proteins unidirectionally around a functional cycle to propel their diverse physical tasks.     

Helical inchworming: a novel translocation mechanism for a ring ATPase

Ring ATPases perform a variety of tasks in the cell. Their function involves complex communication and coordination among the often identical subunits. Translocases in this group are of particular interest as they involve both chemical and mechanical actions in their operation. We study the DNA packaging motor of bacteriophage φ29, and using single-molecule optical tweezers and single-particle cryo-electron microscopy, have discovered a novel translocation mechanism for a molecular motor.     

High-speed atomic force microscopy: Imaging and force spectroscopy

Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding.     

Structural diversity and extreme stability of unimolecular Oxytricha nova telomeric G-quadruplex

Oxytricha nova telomeric DNA contains guanine-rich short-tandem repeat sequences (GGGGTTTT) n and terminates as a single strand at the 3'-end. This single-stranded overhang forms a novel DNA structure, namely, G-quadruplex, comprising four quartets. In this study, we investigated the structures and dynamics of unimolecular Oxytricha nova ( O. nova) telomeric G-quadruplexes by performing single molecule fluorescence resonance energy transfer (FRET) spectroscopy and bulk circular dichroism (CD) measurements. We observed that unimolecular O. nova G-quadruplexes exhibit structural polymorphism according to monovalent cations. In the presence of Na (+), only antiparallel conformation is detected, which was demonstrated in previous studies; however, in the presence of K (+), they fold into two different conformations, a parallel conformation and an antiparallel one different from that induced by Na (+). Furthermore, these G-quadruplexes show extremely high stability in their dynamics when compared with human G-quadruplexes. While human telomeric G-quadruplexes that possess three quartets display fast dynamic behavior (<100 s) at low K (+) concentrations or high temperatures, O. nova G-quadruplexes maintain their conformational state for a long time (>1000 s), even at the lowest K (+) concentration and the highest temperature investigated. This high stability is primarily due to an extra quartet that results in additional cation coordination. In addition to cation coordination, we propose that other factors such as base stacking and the size of the thymine loop may contribute to the stability of O. nova G-quadruplexes; this is based on the fact that the O. nova G-quadruplexes were observed to be more stable than the human ones in the presence of Li (+), which is known to greatly destabilize G-quadruplexes because of imprecise coordination. This extreme stability of four-quartet G-quadruplexes enables telomere protection even in the absence of protective proteins or in the case of abrupt environmental changes, although only a single G-quadruplex structure can be derived from the short single-stranded overhang.     

Moving into the cell: single-molecule studies of molecular motors in complex environments

Much has been learned in the past decades about molecular force generation. Single-molecule techniques, such as atomic force microscopy, single-molecule fluorescence microscopy and optical tweezers, have been key in resolving the mechanisms behind the power strokes, 'processive' steps and forces of cytoskeletal motors. However, it remains unclear how single force generators are integrated into composite mechanical machines in cells to generate complex functions such as mitosis, locomotion, intracellular transport or mechanical sensory transduction. Using dynamic single-molecule techniques to track, manipulate and probe cytoskeletal motor proteins will be crucial in providing new insights.     

Taking it one step at a time in homologous recombination repair

The individual steps in the process of homologous recombination are particularly amenable to analysis by single-molecule imaging and manipulation experiments. Over the past 20 years these have provided a wealth of new information on the DNA transactions that make up this vital process. Exciting progress in developing new tools and techniques to analyze more complex components, dynamic reaction steps and molecular coordination continues at a rapid pace. Here we highlight recent results and indicate some emerging techniques likely to produce the next stage of advanced insight into homologous recombination. In this and related fields the future is bright.     

Atomic Force Microscopy of Asymmetric Membranes from Turtle Erythrocytes

The cell membrane provides critical cellular functions that rely on its elaborate structure and organization. The structure of turtle membranes is an important part of an ongoing study of erythrocyte membranes. Using a combination of atomic force microscopy and single-molecule force spectroscopy, we characterized the turtle erythrocyte membrane structure with molecular resolution in a quasi-native state. High-resolution images both leaflets of turtle erythrocyte membranes revealed a smooth outer membrane leaflet and a protein covered inner membrane leaflet. This asymmetry was verified by single-molecule force spectroscopy, which detects numerous exposed amino groups of membrane proteins in the inner membrane leaflet but much fewer in the outer leaflet. The asymmetric membrane structure of turtle erythrocytes is consistent with the semi-mosaic model of human, chicken and fish erythrocyte membrane structure, making the semi-mosaic model more widely applicable. From the perspective of biological evolution, this result may support the universality of the semi-mosaic model.     

Fast interaction dynamics of G-quadruplex and RGG-rich peptides unveiled in zero-mode waveguides

G-quadruplexes (GQs), a non-canonical form of DNA, are receiving a huge interest as target sites for potential applications in antiviral and anticancer drug treatments. The biological functions of GQs can be controlled by specifically binding proteins known as GQs binding proteins. Some of the GQs binding proteins contain an arginine and glycine-rich sequence known as RGG peptide. Despite the important role of RGG, the GQs-RGG interaction remains poorly understood. By single molecule measurements, the interaction dynamics can be determined in principle. However, the RGG–GQs interaction occurs at micromolar concentrations, making conventional single-molecule experiments impossible with a diffraction-limited confocal microscope. Here, we use a 120 nm zero-mode waveguide (ZMW) nanoaperture to overcome the diffraction limit. The combination of dual-color fluorescence cross-correlation spectroscopy (FCCS) with FRET is used to unveil the interaction dynamics and measure the association and dissociation rates. Our data show that the RGG–GQs interaction is predominantly driven by electrostatics but that a specific affinity between the RGG sequence and the GQs structure is preserved. The single molecule approach at micromolar concentration is the key to improve our understanding of GQs function and develop its therapeutic applications by screening a large library of GQs-targeting peptides and proteins.     

Dramatic effect of single-base mutation on the conformational dynamics of human telomeric G-quadruplex

Guanine-rich DNA sequences can form G-quadruplexes. These four-stranded structures are known to form in several genomic regions and to influence certain biological activities. Sometimes, the instability of G-quadruplexes causes the abnormal biological processes. Mutation is a culprit for the destabilization of G-quadruplexes, but the details of mutated G-quadruplexes are poorly understood. In this article, we investigated the conformational dynamics of single-base mutated human telomeric G-quadruplexes in the presence of K⁺ with single-molecule FRET spectroscopy. We observed that the replacement of single guanine by thymine in a G-track induces various folded structures, i.e. structural polymorphism. Moreover, direct observation of their dynamics revealed that a single-base mutation causes fast unfolding of folded states under physiological conditions. Furthermore, we found that the degree of destabilization varies according to mutation positions. When the central guanine of a G-track is replaced, the G-quadruplexes unfold quickly at any K⁺ concentrations and temperature. Meanwhile, outer-quartet mutated G-quadruplexes have heterogeneous dynamics at intermediate K⁺ concentrations and longstanding folded states at high K⁺ concentrations. Several factors such as base-stacking interaction and K⁺ coordination are responsible for the different dynamics according to the mutation position.     

Single-molecule studies of DNA replisome function

Fast and accurate replication of DNA is accomplished by the interactions of multiple proteins in the dynamic DNA replisome. The DNA replisome effectively coordinates the leading and lagging strand synthesis of DNA. These complex, yet elegantly organized, molecular machines have been studied extensively by kinetic and structural methods to provide an in-depth understanding of the mechanism of DNA replication. Owing to averaging of observables, unique dynamic information of the biochemical pathways and reactions is concealed in conventional ensemble methods. However, recent advances in the rapidly expanding field of single-molecule analyses to study single biomolecules offer opportunities to probe and understand the dynamic processes involved in large biomolecular complexes such as replisomes. This review will focus on the recent developments in the biochemistry and biophysics of DNA replication employing single-molecule techniques and the insights provided by these methods towards a better understanding of the intricate mechanisms of DNA replication.     

Single-molecule studies of riboswitch folding

The folding dynamics of riboswitches are central to their ability to modulate gene expression in response to environmental cues. In most cases, a structural competition between the formation of a ligand-binding aptamer and an expression platform (or some other competing off-state) determines the regulatory outcome. Here, we review single-molecule studies of riboswitch folding and function, predominantly carried out using single-molecule FRET or optical trapping approaches. Recent results have supplied new insights into riboswitch folding energy landscapes, the mechanisms of ligand binding, the roles played by divalent ions, the applicability of hierarchical folding models, and kinetic vs. thermodynamic control schemes. We anticipate that future work, based on improved data sets and potentially combining multiple experimental techniques, will enable the development of more complete models for complex RNA folding processes. This article is part of a Special Issue entitled: Riboswitches.