Single-molecule Force Spectroscopy Research Articles

Chain-Length Dependence of Peptide-Lipid Bilayer Interaction Strength and Binding Kinetics: A Combined Theoretical and Experimental Approach.

Physical interactions between polypeptide chains and lipid membranes underlie critical cellular processes. Yet, despite fundamental importance, key mechanistic aspects of these interactions remain elusive. Bulk experiments have revealed a linear relationship between free energy and peptide chain length in a model system, but does this linearity extend to the interaction strength and to the kinetics of lipid binding? To address these questions, we utilized a combination of coarse-grained molecular dynamics (CG MD) simulations, analytical modeling, and atomic force microscopy (AFM)-based single molecule force spectroscopy. Following previous bulk experiments, we focused on interactions between short hydrophobic peptides (WLn, n = 1, ..., 5) with 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayers, a simple system that probes peptide primary structure effects. Potentials of mean force extracted from CG MD recapitulated the linearity of free energy with the chain length. Simulation results were quantitatively connected to bulk biochemical experiments via a single scaling factor of order unity, corroborating the methodology. Additionally, CG MD revealed an increase in the distance to the transition state, a result that weakens the dependence of the dissociation force on the peptide chain length. AFM experiments elucidated rupture force distributions and, through modeling, intrinsic dissociation rates. Taken together, the analysis indicates a rupture force plateau in the WLn-POPC system, suggesting that the final rupture event involves the last 2 or 3 residues. In contrast, the linear dependence on chain length was preserved in the intrinsic dissociation rate. This study advances the understanding of peptide-lipid interactions and provides potentially useful insights for the design of peptides with tailored membrane-interacting properties.

The condensation of HP1α/Swi6 imparts nuclear stiffness

Biomolecular condensates have emerged as major drivers of cellular organization. It remains largely unexplored, however, whether these condensates can impart mechanical function(s) to the cell. The heterochromatin protein HP1α (Swi6 in Schizosaccharomyces pombe) crosslinks histone H3K9 methylated nucleosomes and has been proposed to undergo condensation to drive the liquid-like clustering of heterochromatin domains. Here, we leverage the genetically tractable S.pombe model and a separation-of-function allele to elucidate a mechanical function imparted by Swi6 condensation. Using single-molecule imaging, force spectroscopy, and high-resolution live-cell imaging, we show that Swi6 is critical for nuclear resistance to external force. Strikingly, it is the condensed yet dynamic pool of Swi6, rather than the chromatin-bound molecules, that is essential to imparting mechanical stiffness. Our findings suggest that Swi6 condensates embedded in the chromatin meshwork establish the emergent mechanical behavior of the nucleus as a whole, revealing that biomolecular condensation can influence organelle and cell mechanics.

A Handle-Free, All-Protein-Based Optical Tweezers Method to Probe Protein Folding-Unfolding Dynamics.

Optical tweezers (OT) have evolved into powerful single molecule force spectroscopy tools to investigate protein folding-unfolding dynamics. To stretch a protein of interest using OT, the protein must be flanked with two double stranded DNA (dsDNA) handles. However, coupling dsDNA handles to the protein is often of low yield, representing a bottleneck in OT experiments. Here, we report a handle-free, all-protein-based OT method for investigating protein folding/unfolding dynamics. In this new method, we employed disordered elastin-like polypeptides (ELPs) as a molecular linker and the mechanically stable cohesin-dockerin (Coh-Doc) pair as the prey-bait system to enable the efficient capture and stretching of individual protein molecules. This novel approach was validated by using model proteins NuG2 and RTX-v, yielding experimental results comparable to those obtained by using the dsDNA handle approach. This new method provides a streamlined and efficient OT approach to investigate the folding-unfolding dynamics of proteins at the single molecule level, thus expanding the toolbox of OT-based single molecule force spectroscopy.

Stick-slip unfolding favors self-association of expanded HTT mRNA.

In Huntington's Disease (HD) and related disorders, expansion of CAG trinucleotide repeats produces a toxic gain of function in affected neurons. Expanded huntingtin (expHTT) mRNA forms aggregates that sequester essential RNA binding proteins, dysregulating mRNA processing and translation. The physical basis of RNA aggregation has been difficult to disentangle owing to the heterogeneous structure of the CAG repeats. Here, we probe the folding and unfolding pathways of expHTT mRNA using single-molecule force spectroscopy. Whereas normal HTT mRNAs unfold reversibly and cooperatively, expHTT mRNAs with 20 or 40 CAG repeats slip and unravel non-cooperatively at low tension. Slippage of CAG base pairs is punctuated by concerted rearrangement of adjacent CCG trinucleotides, trapping partially folded structures that readily base pair with another RNA strand. We suggest that the conformational entropy of the CAG repeats, combined with stable CCG base pairs, creates a stick-slip behavior that explains the aggregation propensity of expHTT mRNA.

Plant-Derived Anti-Human Epidermal Growth Factor Receptor 2 Antibody Suppresses Trastuzumab-Resistant Breast Cancer with Enhanced Nanoscale Binding.

Traditional monoclonal antibodies such as Trastuzumab encounter limitations when treating Human Epidermal Growth Factor Receptor 2 (HER2)-positive breast cancer, particularly in cases that develop resistance. This study introduces plant-derived anti-HER2 variable fragments of camelid heavy chain domain (VHH) fragment crystallizable region (Fc) KEDL(K) antibody as a potent alternative for overcoming these limitations. A variety of biophysical techniques, in vitro assays, and in vivo experiments uncover the antibody's nanoscale binding dynamics with transmembrane HER2 on living cells. Single-molecule force spectroscopy reveals the rapid formation of two robust bonds, exhibiting approximately 50 pN force resistance and bond lifetimes in the second range. The antibody demonstrates a specific affinity for HER2-positive breast cancer cells, including those that are Trastuzumab-resistant. Moreover, in immune-deficient mice, the plant-derived anti-HER2 VHH-FcK antibody exhibits superior antitumor activity, especially against tumors that are resistant to Trastuzumab. These findings underscore the plant-derived antibody's potential as an impactful immunotherapeutic strategy for treating Trastuzumab-resistant HER2-positive breast cancer.

Direct investigations of interactions between nucleolins and aptamers on pancreatic cancer and normal cells by atomic force microscopy

Nucleolin is overexpressed on the surface of pancreatic cancer cells and are regarded as the remarkable therapeutic target. Aptamers are capable of binding the external domain of nucleolin on the cell surface with high affinity and specificity. But nucleolin has not been localized on pancreatic cancer cells at very high spatial resolution, and the interactions between nucleolin and aptamers have not been investigated at very high force resolution level. In this work, nucleolin was localized on pancreatic cancer and normal cells by aptamers (9FU-AS1411-NH2, AS1411-NH2 and CRONH2) in Single Molecule Recognition Imaging mode of Atomic Force Microscopy. There are plenty of nucleolin on the surfaces of pancreatic cancer cells (area percentage about 5 %), while there are little nucleolin on the surfaces of normal cells. The interactions between three types of aptamers and nucleolins on the surfaces of pancreatic cancer cells were investigated by Single Molecule Force Spectroscopy. The unbinding forces of nucleolins-(9FU-AS1411-NH2) are larger than nucleolins-(AS1411-NH2). The dissociation activation energy on nucleolin-(9FU-AS1411-NH2) is higher than nucleolin-(AS1411-NH2), which indicates that the former complex is more stable and harder to dissociate than the later complex. There are no unbinding forces between nucleolin and CRONH2. All these demonstrate that nucleolin was localized on pancreatic cancer and normal cells at single molecule level quantitatively, and the interactions (unbinding forces and kinetics) between nucleolin and aptamers were studied at picoNewton level. The approaches and results of this work will pave new ways in the investigations of nucleolin and aptamers, and will also be useful in the studies on other proteins and their corresponding aptamers.

Understanding the Mechanochemistry of Mechano-Radicals in Self-Growth Materials by Single-Molecule Force Spectroscopy.

Recent research on mechano-radicals has provided valuable insights into self-growth and adaptive responsive materials. Typically, mechanophores must remain inert in the absence of force but respond quickly to external tension before other linkages within the polymer network. Azo compounds exhibit promising combinations of mechanical stability and force-triggered reactivity, making them widely used as mechano-radicals in force-responsive materials. However, the activation conditions and behavior of azo compounds have yet to be quantitatively explored. In this study, we investigated the mechanical strength of three azo compounds using single-molecule force spectroscopy. Our results revealed that these compounds exhibit rupture forces ranging from ~500 to 1000 pN, at a loading rate of 3×104 pN s-1. Importantly, these mechanophores demonstrate distinct kinetic properties. Their unique mechanical attributes enable azo bond scission and free radical generation before causing major polymer backbone damage of entire material during polymer network deformation. This fundamental understanding of mechanophores holds significant promise for the development of self-growth materials and their related applications.

Click Chemistry-Based Force Spectroscopy Revealed Enhanced Binding Dynamics of Phosphorylated HMGB1 to Cisplatin-DNA.

Cisplatin, a cornerstone in cancer chemotherapy, is known for its DNA-binding capacity and forms lesions that lead to cancer cell death. However, the repair of these lesions compromises cisplatin's effectiveness. This study investigates how phosphorylation of HMGB1, a nuclear protein, modifies its binding to cisplatin-modified DNA (CP-DNA) and thus protects it from repair. Despite numerous methods for detecting protein-DNA interactions, quantitative approaches for understanding their molecular mechanism remain limited. Here, we applied click chemistry-based single-molecule force spectroscopy, achieving high-precision quantification of the interaction between phosphorylated HMGB1 and CP-DNA. This method utilizes a synergy of click chemistry and enzymatic ligation for precise DNA-protein immobilization and interaction in the system. Our results revealed that HMGB1 binds to CP-DNA with a significantly high rupture force of ∼130 pN, stronger than most natural DNA-protein interactions and varying across different DNA sequences. Moreover, Ser14 is identified as the key phosphorylation site, enhancing the interaction's kinetic stability by 35-fold. This increase in stability is attributed to additional hydrogen bonding suggested by molecular dynamics (MD) simulations. Our findings not only reveal the important role of phosphorylated HMGB1 in potentially improving cisplatin's therapeutic efficacy but also provide a precise method for quantifying protein-DNA interactions.

Heterogeneous and Cooperative Rupture of Histidine-Ni2+ Metal-Coordination Bonds on Rationally Designed Protein Templates.

Metal-coordination bonds, a highly tunable class of dynamic noncovalent interactions, are pivotal to the function of a variety of protein-based natural materials and have emerged as binding motifs to produce strong, tough, and self-healing bioinspired materials. While natural proteins use clusters of metal-coordination bonds, synthetic materials frequently employ individual bonds, resulting in mechanically weak materials. To overcome this current limitation, we rationally designed a series of elastin-like polypeptide templates with the capability of forming an increasing number of intermolecular histidine-Ni2+ metal-coordination bonds. Using single-molecule force spectroscopy and steered molecular dynamics simulations, we show that templates with three histidine residues exhibit heterogeneous rupture pathways, including the simultaneous rupture of at least two bonds with more-than-additive rupture forces. The methodology and insights developed improve our understanding of the molecular interactions that stabilize metal-coordinated proteins and provide a general route for the design of new strong, metal-coordinated materials with a broad spectrum of dissipative time scales.

Effects of Proline on Internal Friction in Simulated Folding Dynamics of Several Alanine-Based α-Helical Peptides.

We have studied in silico the effect of proline, a model cosolvent, on local and global friction coefficients in (un)folding of several typical alanine-based α-helical peptides. Local friction is related to dwell times of a single, ensemble-averaged hydrogen bond (HB) within each peptide. Global friction is related to energy dissipated in a series of configurational changes of each peptide experienced by increasing the number of HBs during folding. Both of these approaches are important in relation to future atomic force microscopic-based measurements of internal friction via force-clamp single-molecule force spectroscopy. Molecular dynamics (MD) simulations for six peptides, namely, ALA5, ALA8, ALA15, ALA21, (AAQAA)3, and H2N-GN(AAQAA)2G-COONH2, have been conducted at 2 and 5 M proline solutions in water. Using previously obtained MD data for these peptides in pure water as well as upgraded theoretical models, we obtained variations of local and global internal friction coefficients as a function of solution viscosity. The results showed the substantial role of proline in stabilizing the folded state and slowing the overall folding dynamics. Consequently, larger friction coefficients were obtained at larger viscosities. The local and global internal friction, i.e., respective, friction coefficients approximated to zero viscosity, was also obtained. The evolution of friction coefficients with viscosity was weakly dependent on the number of concurrent folding pathways but was rather dominated by a stabilizing effect of proline on the folded states. Obtained values of local and global internal friction showed qualitatively similar results and a clear dependency on the structure of the studied peptide.

Engineering an artificial catch bond using mechanical anisotropy

Catch bonds are a rare class of protein-protein interactions where the bond lifetime increases under an external pulling force. Here, we report how modification of anchor geometry generates catch bonding behavior for the mechanostable Dockerin G:Cohesin E (DocG:CohE) adhesion complex found on human gut bacteria. Using AFM single-molecule force spectroscopy in combination with bioorthogonal click chemistry, we mechanically dissociate the complex using five precisely controlled anchor geometries. When tension is applied between residue #13 on CohE and the N-terminus of DocG, the complex behaves as a two-state catch bond, while in all other tested pulling geometries, including the native configuration, it behaves as a slip bond. We use a kinetic Monte Carlo model with experimentally derived parameters to simulate rupture force and lifetime distributions, achieving strong agreement with experiments. Single-molecule FRET measurements further demonstrate that the complex does not exhibit dual binding mode behavior at equilibrium but unbinds along multiple pathways under force. Together, these results show how mechanical anisotropy and anchor point selection can be used to engineer artificial catch bonds.

A Thermally Stable SO2-Releasing Mechanophore: Facile Activation, Single-Event Spectroscopy, and Molecular Dynamic Simulations.

Polymers that release small molecules in response to mechanical force are promising candidates as next-generation on-demand delivery systems. Despite advancements in the development of mechanophores for releasing diverse payloads through careful molecular design, the availability of scaffolds capable of discharging biomedically significant cargos in substantial quantities remains scarce. In this report, we detail a nonscissile mechanophore built from an 8-thiabicyclo[3.2.1]octane 8,8-dioxide (TBO) motif that releases one equivalent of sulfur dioxide (SO2) from each repeat unit. The TBO mechanophore exhibits high thermal stability but is activated mechanochemically using solution ultrasonication in either organic solvent or aqueous media with up to 63% efficiency, equating to 206 molecules of SO2 released per 143.3 kDa chain. We quantified the mechanochemical reactivity of TBO by single-molecule force spectroscopy and resolved its single-event activation. The force-coupled rate constant for TBO opening reaches ∼9.0 s-1 at ∼1520 pN, and each reaction of a single TBO domain releases a stored length of ∼0.68 nm. We investigated the mechanism of TBO activation using ab initio steered molecular dynamic simulations and rationalized the observed stereoselectivity. These comprehensive studies of the TBO mechanophore provide a mechanically coupled mechanism of multi-SO2 release from one polymer chain, facilitating the translation of polymer mechanochemistry to potential biomedical applications.

The application of single-molecule optical tweezers to study disease-related structural dynamics in RNA.

RNA, a dynamic and flexible molecule with intricate three-dimensional structures, has myriad functions in disease development. Traditional methods, such as X-ray crystallography and nuclear magnetic resonance, face limitations in capturing real-time, single-molecule dynamics crucial for understanding RNA function. This review explores the transformative potential of single-molecule force spectroscopy using optical tweezers, showcasing its capability to directly probe time-dependent structural rearrangements of individual RNA molecules. Optical tweezers offer versatility in exploring diverse conditions, with the potential to provide insights into how environmental changes, ligands and RNA-binding proteins impact RNA behaviour. By enabling real-time observations of large-scale structural dynamics, optical tweezers emerge as an invaluable tool for advancing our comprehension of RNA structure and function. Here, we showcase their application in elucidating the dynamics of RNA elements in virology, such as the pseudoknot governing ribosomal frameshifting in SARS-CoV-2.

Mechanistic Insight into the Mechanical Unfolding of the Integral Membrane Diacylglycerol Kinase.

The essential forces stabilizing membrane proteins and governing their folding and unfolding are difficult to decipher. Single-molecule atomic force spectroscopy mechanically unfolds individual membrane proteins and quantifies their dynamics and energetics. However, it remains challenging to structurally assign unfolding intermediates precisely and to deduce dominant interactions between specific residues that facilitate either the localized stabilization of these intermediates or the global assembly of membrane proteins. Here, we performed force spectroscopy experiments and multiscale molecular dynamics simulations to study the unfolding pathway of diacylglycerol kinase (DGK), a small trimeric multispan transmembrane enzyme. The remarkable agreement between experiments and simulations allowed precise structural assignment and interaction analysis of unfolding intermediates, bypassing existing limitations on structural mapping, and thus provided mechanistic explanations for the formation of these states. DGK unfolding was found to proceed with structural segments varying in size that do not correlate with its secondary structure. We identified intermolecular side-chain packing interactions as one of the major contributions to the stability of unfolding intermediates. Mutagenesis creating packing defects induced a dramatic decrease in the mechano-stability of corresponding intermediates and also in the thermo-stability of DGK trimer, in good agreement with predictions from simulations. Hence, the molecular determinants of the mechano- and thermo-stability of a membrane protein can be identified at residue resolution. The accurate structural assignment established and microscopic mechanism revealed in this work may substantially expand the scope of single-molecule studies of membrane proteins.

Single-Molecule Investigation of the Binding Interface Stability of SARS-CoV-2 Variants with ACE2.

The SARS-CoV-2 pandemic spurred numerous research endeavors to comprehend the virus and mitigate its global severity. Understanding the binding interface between the virus and human receptors is pivotal to these efforts and paramount to curbing infection and transmission. Here we employ atomic force microscopy and steered molecular dynamics simulation to explore SARS-CoV-2 receptor binding domain (RBD) variants and angiotensin-converting enzyme 2 (ACE2), examining the impact of mutations at key residues upon binding affinity. Our results show that the Omicron and Delta variants possess strengthened binding affinity in comparison to the Mu variant. Further, using sera from individuals either vaccinated or with acquired immunity following Delta strain infection, we assess the impact of immunity upon variant RBD/ACE2 complex formation. Single-molecule force spectroscopy analysis suggests that vaccination before infection may provide stronger protection across variants. These results underscore the need to monitor antigenic changes in order to continue developing innovative and effective SARS-CoV-2 abrogation strategies.

High-throughput virtual search of small molecules for controlling the mechanical stability of human CD4

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods, may have important implications in drug development and discovery. Here, we present the first approach that employs computational High-Throughput Virtual Screening (HTVS) and Molecular Docking to search for small-molecules in chemical libraries that function as mechano-regulators of the stability of human CD4, receptor of HIV-1. Using single-molecule force spectroscopy we prove that these small-molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, i.e., mechano-regulation, paving the way towards mechanopharmacology.

Nucleosome Dynamics Derived at the Single-Molecule Level Bridges Its Structures and Functions.

Nucleosome, the building block of chromatin, plays pivotal roles in all DNA-related processes. While cryogenic-electron microscopy (cryo-EM) has significantly advanced our understanding of nucleosome structures, the emerging field of single-molecule force spectroscopy is illuminating their dynamic properties. This technique is crucial for revealing how nucleosome behavior is influenced by chaperones, remodelers, histone variants, and post-translational modifications, particularly in their folding and unfolding mechanisms under tension. Such insights are vital for deciphering the complex interplay in nucleosome assembly and structural regulation, highlighting the nucleosome's versatility in response to DNA activities. In this Perspective, we aim to consolidate the latest advancements in nucleosome dynamics, with a special focus on the revelations brought forth by single-molecule manipulation. Our objective is to highlight the insights gained from studying nucleosome dynamics through this innovative approach, emphasizing the transformative impact of single-molecule manipulation techniques in the field of chromatin research.

Mechanistic Analysis of Riboswitch Ligand Interactions Provides Insights into Pharmacological Control over Gene Expression.

Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ 1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis ( Tte )-PreQ 1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ 1 in six structurally and mechanistically diverse PreQ 1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms.

Mechanistic insights into the C-type lectin receptor CLEC12A-mediated immune recognition of monosodium urate crystal

CLEC12A, a member of the C-type lectin receptor family involved in immune homeostasis, recognizes MSU crystals released from dying cells. However, the molecular mechanism underlying the CLEC12A-mediated recognition of MSU crystals still remains unclear. Herein, we reported the crystal structure of the human CLEC12A-CTLD and identified a unique “basic patch” site on CLEC12A-CTLD that is necessary for the binding of MSU crystals. Meanwhile, we determined the interaction strength between CLEC12A-CTLD and MSU crystals using single-molecule force spectroscopy. Furthermore, we found that CLEC12A clusters at cell membrane and seems to serve as an internalizing receptor of MSU crystals. Altogether, these findings provide mechanistic insights for understanding the molecular mechanisms underlying the interplay between CLEC12A and MSU crystals.

Special contribution of atomic force microscopy in cell death research

Abstract Cell death is an important life activity in individual development. Changes in morphological and mechanical properties during cell death are crucial to identify the modes of cell death. However, due to technical limitations, little is known about these characteristics. The emergence of atomic force microscopy (AFM), a nanoscale research tool that integrates imaging and mechanical measurement functions, provides new insights into our understanding of cell death. Based on a brief introduction to the structure, principle, and working modes of AFM, this article elaborates on the contribution of AFM in cell death to detect morphological and mechanical properties, especially in apoptotic cells. Meanwhile, the potential of AFM in distinguishing different cell death modes and visualizing membrane pores (medicated by apoptosis and pyroptosis) is illustrated. In addition, this article states that using single-molecule force spectroscopy by AFM to study the mechanical and adhesive properties of cell death-related molecules. Finally, we discuss the challenges facing and further perspective of AFM.