Single Molecule AFM Force Spectroscopy Research Articles

Engineering the Mechanical Stability of a Therapeutic Complex between Affibody and Programmed Death-Ligand 1 by Anchor Point Selection.

Protein-protein complexes can vary in mechanical stability depending on the direction from which force is applied. Here, we investigated the mechanical stability of a complex between a binding scaffold called Affibody and an immune checkpoint protein Programmed Death-Ligand 1 (PD-L1). We used AFM single-molecule force spectroscopy with bioorthogonal clickable peptide handles, shear stress bead adhesion assays, molecular modeling, and steered molecular dynamics (SMD) to understand the pulling point dependency of the mechanostability of the Affibody:(PD-L1) complex. We observed a wide range of rupture forces depending on the anchor point. Pulling from residue #22 on Affibody generated an intermediate state attributed to partially unfolded PD-L1, while pulling from Affibody's N-terminus generated a force-activated catch bond. Pulling from residue #22 or #47 on Affibody generated high rupture forces, with the complex breaking at up to ∼190 pN under loading rates of ∼104-105 pN/s, representing a ∼4-fold increase as compared with low-force N-terminal pulling. SMD simulations showed relative tendencies in rupture forces that were consistent with experiments and, through visualization of force propagation networks, provided mechanistic insights. These results demonstrate how the mechanical properties of protein-protein interfaces can be controlled by informed choice of site-specific bioconjugation points within molecules, with implications for optimal bioconjugation strategies in drug delivery vehicles.

Engineering the Mechanical Stability of a Therapeutic Affibody/PD-L1 Complex by Anchor Point Selection.

Protein-protein complexes can vary in mechanical stability depending on the direction from which force is applied. Here we investigated the anisotropic mechanical stability of a molecular complex between a therapeutic non-immunoglobulin scaffold called Affibody and the extracellular domain of the immune checkpoint protein PD-L1. We used a combination of single-molecule AFM force spectroscopy (AFM-SMFS) with bioorthogonal clickable peptide handles, shear stress bead adhesion assays, molecular modeling, and steered molecular dynamics (SMD) simulations to understand the pulling point dependency of mechanostability of the Affibody:(PD-L1) complex. We observed diverse mechanical responses depending on the anchor point. For example, pulling from residue #22 on Affibody generated an intermediate unfolding event attributed to partial unfolding of PD-L1, while pulling from Affibody's N-terminus generated force-activated catch bond behavior. We found that pulling from residue #22 or #47 on Affibody generated the highest rupture forces, with the complex breaking at up to ~ 190 pN under loading rates of ~104-105 pN/sec, representing a ~4-fold increase in mechanostability as compared with low force N-terminal pulling. SMD simulations provided consistent tendencies in rupture forces, and through visualization of force propagation networks provided mechanistic insights. These results demonstrate how mechanostability of therapeutic protein-protein interfaces can be controlled by informed selection of anchor points within molecules, with implications for optimal bioconjugation strategies in drug delivery vehicles.

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.

Mapping Mechanostable Pulling Geometries of a Therapeutic Anticalin/CTLA-4 Protein Complex.

We used single-molecule AFM force spectroscopy (AFM-SMFS) in combination with click chemistry to mechanically dissociate anticalin, a non-antibody protein binding scaffold, from its target (CTLA-4), by pulling from eight different anchor residues. We found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in koff by 2–3 orders of magnitude over a force range of 50–200 pN. Five of the six internal anchor points gave rise to complexes significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1–10 nN s–1. Anisotropic network modeling and molecular dynamics simulations helped to explain the geometric dependency of mechanostability. These results demonstrate that optimization of attachment residue position on therapeutic binding scaffolds can provide large improvements in binding strength, allowing for mechanical affinity maturation under shear stress without mutation of binding interface residues.

Mapping Mechanostable Pulling Geometries of Protein-Ligand Complexes

Anticalin is a non-immunoglobulin protein scaffold with potential as an alternative to monoclonal antibodies for nanoparticle-based drug delivery to cells displaying cytotoxic T-lymphocyte antigen 4 (CTLA-4). In this context, one limiting factor is the resistance of the anticalin:CTLA-4 complex to mechanical forces exerted by fluid shear stress. Here, we used single-molecule AFM force spectroscopy to screen residues along the anticalin backbone and determine the optimal pulling point that achieves maximum mechanical stability of the anticalin:CTLA-4 complex. We used non-canonical amino acid incorporation by amber suppression in the anticalin combined with click chemistry to attach an Fgβ peptide at internal residues of the anticalin. We then used the Fgβ peptide as a handle to mechanically dissociate anticalin from CTLA-4 by applying tension at 8 different anchor residues, and measure the unbinding energy landscape for each pulling geometry. We found that pulling from amino acid position 60 on the anticalin resulted in ∼100% higher mechanical stability of the complex as compared with either the N- or C-terminus. Molecular dynamics (MD) simulations using the coarse-grained Martini force field showed strong agreement with experiments and help explain the mechanisms underlying the geometric dependency of mechanical stability in this therapeutic molecular complex. These results demonstrate that the mechanical stability of receptor-ligand complexes can be optimized by controlling the loading geometry without making any changes to the binding interface.

Force-Induced Unravelling of DNA Origami.

The mechanical properties of DNA nanostructures are of widespread interest as applications that exploit their stability under constant or intermittent external forces become increasingly common. We explore the force response of DNA origami in comprehensive detail by combining AFM single molecule force spectroscopy experiments with simulations using oxDNA, a coarse-grained model of DNA at the nucleotide level, to study the unravelling of an iconic origami system: the Rothemund tile. We contrast the force-induced melting of the tile with simulations of an origami 10-helix bundle. Finally, we simulate a recently proposed origami biosensor, whose function takes advantage of origami behavior under tension. We observe characteristic stick-slip unfolding dynamics in our force-extension curves for both the Rothemund tile and the helix bundle and reasonable agreement with experimentally observed rupture forces for these systems. Our results highlight the effect of design on force response: we observe regular, modular unfolding for the Rothemund tile that contrasts with strain-softening of the 10-helix bundle which leads to catastropic failure under monotonically increasing force. Further, unravelling occurs straightforwardly from the scaffold ends inward for the Rothemund tile, while the helix bundle unfolds more nonlinearly. The detailed visualization of the yielding events provided by simulation allows preferred pathways through the complex unfolding free-energy landscape to be mapped, as a key factor in determining relative barrier heights is the extensional release per base pair broken. We shed light on two important questions: how stable DNA nanostructures are under external forces and what design principles can be applied to enhance stability.

Calcium Dependent Interaction Between N2A-Halo and F-Actin: A Single Molecule Study

In the last two decades, interactions between titin's I-band region and actin have been widely investigated for skeletal and cardiac isoforms. Previous studies suggested a calcium-dependent interaction between titin and actin. However, previous in vitro motility studies did not quantify the strength of interaction between titin's N2A region and actin in the presence of calcium. Here, we used AFM single molecule force spectroscopy (SMFS) to probe the interaction strength between a recombinant N2A-HALO construct and actin filaments (F-actin), in the presence (pCa = 4.0) and absence of calcium. The N2A-Halo construct consisted of the N2A region of titin (Ig80-IS-Ig81-Ig82-Ig83, where IS is the insertion sequence) with a C-terminal Halo-tag. The construct was immobilized on an AFM cantilever tip via its N-terminal cysteines. The interaction strength was determined by probing the molecule during multiple approach-retraction cycles at physiological pulling rates (300-1000 nm/sec) over various locations on a substrate functionalized with F-actin stabilized on a lipid bilayer with ATP. Preliminary data show that both the yield and strength of N2A-Halo interactions with F-actin nearly doubled in the presence of Ca2+, with multiple peaks in the rupture force distribution at 50 ± 25 pN, 75 ± 10 pN, and 100 ± 25 pN. The force extension curves also showed different rupture lengths, indicating multiple actin binding sites. No significant interactions occurred between N2A constructs and the charged lipid bi-layer surface in the absence of F-actin. These experiments show that calcium increases the interaction probability and binding strength between N2A-Halo and F-actin and that the binding strength between N2A titin and F-actin is nearly 4 times greater than previously reported for PEVK titin - F-actin interactions. These results support a physiological role for titin in active muscle contraction and force enhancement.

Increasing evidence of mechanical force as a functional regulator in smooth muscle myosin light chain kinase.

Mechanosensitive proteins are key players in cytoskeletal remodeling, muscle contraction, cell migration and differentiation processes. Smooth muscle myosin light chain kinase (smMLCK) is a member of a diverse group of serine/threonine kinases that feature cytoskeletal association. Its catalytic activity is triggered by a conformational change upon Ca2+/calmodulin (Ca2+/CaM) binding. Due to its significant homology with the force-activated titin kinase, smMLCK is suspected to be also regulatable by mechanical stress. In this study, a CaM-independent activation mechanism for smMLCK by mechanical release of the inhibitory elements is investigated via high throughput AFM single-molecule force spectroscopy. The characteristic pattern of transitions between different smMLCK states and their variations in the presence of different substrates and ligands are presented. Interaction between kinase domain and regulatory light chain (RLC) substrate is identified in the absence of CaM, indicating restored substrate-binding capability due to mechanically induced removal of the auto-inhibitory regulatory region.

Mechanically Unfolded Titin Immunoglobulin Domains Refold Faster and More Accurately in Presence of Chaperone Alpha-B-Crystallin

The spring region of the giant muscle protein titin contains many immunoglobulin-like (Ig) domains, which unfold and refold under low (physiological) stretch forces. Small heat shock proteins such as alphaB-crystallin (aBC) translocate under physiological or pathological stress to the titin springs. To better understand this protective function we studied the unfolding-refolding behavior of an 8-Ig-domain titin construct (I91)8 by single-molecule AFM force spectroscopy, in the absence/presence of recombinant aBC (pH7; pH6; or pH5) or ‘control’ protein of similar size. Titin Ig domains were unfolded at 175 pN constant force applied for a variable “denature” time (tD), then the force was set to zero for a variable “quench” time (tQ) to allow for domain refolding, and finally a “probe” pulse (175 pN; tP = 5 s) was applied to test how many domains had refolded. Interestingly, Ig domain unfolding kinetics were little affected by aBC. However, upon lowering pH from 7 to 6, the refolded fraction (number of refolded Ig domains during tQ indexed to number of unfolded Ig domains during tD) decreased slightly, indicating domain misfolding. At pH5, the refolded fraction dropped by half and ∼50% of titin Ig domains showed misfolding events, an effect independent of tD (variation, 2 - 40 s). Importantly, aBC (10 µM or 20 µM) normalized the refolded fraction to values observed at pH7, whereas control protein had no such protective effect. The refolded fraction depended strongly on tQ (variation, 0.5 - 10 s), under all experimental conditions. Ig domain refolding kinetics were greatly slowed by lowering pH from 7 to 5, as quantified on refolded fraction vs. tQ plots, on which means were fit by simple exponentials. Again, aBC normalized the refolding kinetics to those observed at pH7 in the absence of aBC, whereas control protein had no such effect. We conclude that aBC speeds up titin Ig domain refolding (novel foldase activity!) and protects from domain misfolding, especially under acidic stress, which is frequently encountered in muscle cells.

Self-Assembly of Full-Size Amyloid Beta 40 Proteins in Dimers

Even with growing evidence for the involvement of protein oligomeric nano-assemblies in the development of protein misfolding diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD) among others, very limited knowledge exists regarding the molecular mechanisms underlying these processes. This is primarily because the protein aggregates are stabilized by weak interactions that are typically transient in nature and difficult to measure. Therefore, only mixtures of aggregates with different morphologies have been studied thus far; in turn, it is unknown how the aggregation process is initiated and how the growth of oligomers progresses. On the other hand, computational approaches can provide detailed descriptions of how monomers bind to the segments of fibrils. However, these powerful simulations, which provide the structure and dynamics of aggregates at the atomic level, require knowledge of the initial nanoassembly structure; in turn, computational analysis primarily models the elongation process of fibrils. Thus, available computational approaches can be applied to modeling the assembly of oligomers if the structure of the oligomer is known. Herein, we have conducted in silico simulations of Aβ40 monomer assembly in dimers. The simulated structures were validated by comparing Monte Carlo pulling simulations with experimental data from single molecule AFM force spectroscopy enabling us to identify dimers structures rupture of which were in good agreement with the experimental results. One important observation from our long time-scale MD simulations is that the dimer does not have the extended beta-structures observed in fibrils. Rather, it remains largely unstructured and is stabilized by hydrophobic interaction and local hydrogen bonds. The results suggest that the Aβ40 aggregation is a complex process in which the monomer structure depends on the aggregate size.

Molecular Tools for Advanced Single-Molecule Studies

Specific attachment and tethering strategies are of utmost importance to study single biomolecules, e.g. in force and fluorescence spectroscopy. To this end we implement and improve molecular tools for use in e.g. AFM-based Single-Molecule Force Spectroscopy (SMFS).The biotin:(Strept)avidin interaction inevitably comes to mind, whenever detection, trapping or purification of biomolecules is desired. Derived thereof, the Strep-Tag II peptide/Strep-Tactin pair eliminates the necessity of biotin-labelling when working with protein specimens. As for (Strept)avidin, the tetravalency in Strep-Tactin is unfavourable for single-molecule applications. We therefore developed a monovalent Strep-Tactin featuring well-defined binding geometry and stoichiometry, yet unaltered affinity towards Strep-Tagged proteins. A unique Cysteine allows for selective immobilization or fluorescence labelling of this construct. We exploited the mechanical properties of the Strep-Tag II:monovalent Strep-Tactin interaction utilizing AFM-based single-molecule force spectroscopy. Rupture forces up to 200 pN are observed, which is comparable to biotin:(Strept)avidin unbinding. The applicability of the system in various force spectroscopy settings to study proteins of interest is evident. In addition, monovalent Strep-Tactin can be considered a precision tool that can replace (Strept)avidin in various applications, especially in mechanically demanding environments and when utilization of the genetically encoded Strep-Tag II is preferential to biotin-labelling.We utilize this novel tethering strategy to for example characterize force-sensing kinases, like Myosin Light Chain Kinase and Focal Adhesion Kinase in AFM SMFS experiments.

Posttranslational Modification of Titin Domains as a Main Regulator of Myocardial Stiffness

The giant protein titin is responsible for the elasticity of striated muscle cells and engages in both mechanical and signaling functions of the heart. TTN, which encodes titin, is a major human disease gene. Mutations in the myosin-bound part of titin account for a large share of familial cases of dilated cardiomyopathy. The elasticity of cardiac titin, which resides in the molecule's I-band region, is regulated by various means, including titin-isoform switching, phosphorylation of unique spring elements by different kinases, and oxidative stress-related alterations to titin. In failing human hearts, titin is hypo-phosphorylated, which appears to be due in part to a phosphorylation deficit at the cardiac-specific N2-B unique sequence and likely contributes to a pathologically increased (titin-based) diastolic stiffness. A recent focus has been the regulation of the mechanical properties of the immunoglobulin-like (Ig-)domains in the titin spring segment. New evidence suggests that a low but non-negligible number of Ig-domains unfolds-refolds within the physiological sarcomere-length range. These Ig-domains are targeted via an oxidative stress-related mechanism recently identified by single-molecule AFM force spectroscopy. If unfolded, the Ig-domains expose cryptic cysteines, which in the presence of oxidized glutathione can become S-glutathionylated. The presence of mixed disulfide(s) with glutathione weakens the mechanical stability of the unfolded Ig-domains and prevents their refolding, thus reducing titin stiffness. Incubation of stretched (skinned) human cardiomyocytes with oxidized glutathione significantly lowered their passive tension, and the effect was fully reversible on incubation with a reductant. In experimental mouse hearts exposed to oxidative stress (0.1 mM H2O2), I-band Ig-domains of titin were identified as preferential targets of oxidation using ICAT labeling/mass spectrometry. These findings establish that disrupted Ig-folding-unfolding dynamics can cause sustained but reversible changes to titin elasticity, which may also be relevant in heart disease.

Oxidative Stress Regulates Titin Elasticity by Affecting Ig-Domain Stability

Background: The elasticity of titin is regulated through several mechanisms, including isoform switching and phosphorylation of unique spring elements (N2-Bus, PEVK). However, the titin springs consist mainly of immunoglobulin-like (Ig) domains, which are centrally involved in the molecular mechanism of titin elasticity. Passive force-regulating mechanisms targeting the Ig-domains of titin have not been described.Methods and Results: We have elucidated a novel oxidative stress-related mechanism regulating muscle elasticity by altering the stability of titin-Ig domains (Alegre-Cebollada et al., Cell. 2014;156:1235-46). Using single-molecule AFM force spectroscopy, force measurements of isolated skinned human myocytes, and redox proteomics, we show that I-band Ig-domains of titin are weakened by oxidative modification of cryptic cysteines. We demonstrate that mechanical unfolding of these Ig domains exposes hidden cysteines, which now become accessible to disulfide bonding or S-glutathionylation in the presence of millimolar concentrations of oxidized glutathione (GSSG). In the AFM experiments, the cysteines of unfolded titin-Ig domains preferentially formed mixed disulfides with glutathione, which prevented the refolding of these domains. Oxidation by GSSG substantially reduced the passive tension of stretched human myocytes, and the effect was fully reversible with the incubation of reduced glutathione. Exposing perfused mouse hearts to oxidative stress (0.1 mM H2O2) revealed that Ig-domains from I-band titin are preferential targets of oxidation, as monitored using ICAT labeling/mass spectrometry.Conclusions: Titin elasticity in striated muscle is modulated by oxidative stress through reversible weakening of Ig-domain stability via S-glutathionylation of buried cysteines. These titin Ig domains could also represent individual mechanosensors, whose mechanical properties determine mechano-chemical signaling processes in stressed myocytes.

Progress in measuring biophysical properties of membrane proteins with AFM single-molecule force spectroscopy

Membrane proteins are crucial in cell physiological activities and are the targets for most drugs. Thus, investigating the behaviors of membrane proteins not only provide deeper insights into cell function, but also help disease treatment and drug development. Atomic force microscopy is a unique tool for investigating the structure of membrane proteins. It can both image the morphology of single native membrane proteins with high resolution and, via single-molecule force spectroscopy (SMFS), directly measure their biophysical properties during molecular physiological activities such as ligand binding and protein unfolding. In the context of molecular biomechanics, SMFS has been successfully used to understand the structure and function of membrane proteins, complementing the static three-dimensional structures of proteins obtained by X-ray crystallography. Here, based on the authors’ antigen-antibody binding force measurements in clinical tumor cells, the principle and method of SMFS is discussed, the progress in using SMFS to characterize membrane proteins is summarized, and challenges for SMFS are presented.

Molecular Mechanisms of Misfolding of Amyloid Peptides

The current model for the development of Alzheimer's, Parkinson's, Huntington's, prion, and other neurodegenerative diseases involves protein misfolding as the early step followed by spontaneous aggregation, with specific proteins identified as the primary initiators for disease development. Therefore, elucidating the properties of the disease-prone misfolded states, understanding the mechanism of their formation, and identification of their most toxic forms will open prospects for the development of early diagnostics and specific therapeutics for these diseases. We have developed single molecule AFM force spectroscopy (SMFS) experimental approach enabling us to probe interprotein interactions and to identify those interactions that correspond to misfolded protein states. Using SMFS, we have discovered that the misfolded dimers are very stable and have a lifetime in a second time scale. Such a long lifetime of dimers suggests that the formation of dimers is the mechanism by which the protein misfolded state is stabilized. We hypothesize that the formation of highly stable misfolded dimers is a critical step in the entire process of the peptide self-assembly into aggregates. The Molecular Dynamics (MD) simulation performed at the μs timescale demonstrated that isolated non-structured monomer upon approaching to each other changed dramatically their initial conformation and formed dimers with antiparallel beta-sheet structures. Steered MD approach showed that the dimers dissociated cooperatively resulting in a sharp rupture peak corresponding to breakage of the beta-sheet structure. Altogether, the SMFS experimental study and computational analysis revealed a critical role of the interpetide interaction in the misfolding process and highlighting the key role of the dimerization in the amyloid aggregation process. Acknowledgements: Grants from NIH (5R01 GM096039-02) and NSF (EPS-1004094).

Single-Molecule AFM Force Spectroscopy Reveals the Difference in the Folding Patterns between Amyloid β 40 and 42 Within Dimers

OPEN SUBJECT AREAS: SINGLE-MOLECULE BIOPHYSICS ALZHEIMER’S DISEASE Received 26 July 2013 Accepted 20 September 2013 Published 7 October 2013 Correspondence and requests for materials should be addressed to Y.L.L. (ylyubchenko@ unmc.edu) Mechanism of amyloid b2protein dimerization determined using single2molecule AFM force spectroscopy Zhengjian Lv 1 , Robin Roychaudhuri 2 , Margaret M. Condron 2 , David B. Teplow 2,3 & Yuri L. Lyubchenko 1 Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198, United States, 2 Department of Neurology, 3 Brain Research Institute and Molecular Biology Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, United States. Ab42 and Ab40 are the two primary alloforms of human amyloid b2protein (Ab). The two additional C2terminal residues of Ab42 result in elevated neurotoxicity compared with Ab40, but the molecular mechanism underlying this effect remains unclear. Here, we used single2molecule force microscopy to characterize interpeptide interactions for Ab42 and Ab40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Ab42 and Ab40 monomers within dimers. Although the sequence difference between the two peptides is at the C2termini, the N2terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N2terminal was considered as disordered segment with no effect on the Ab peptide aggregation. These novel properties of Ab proteins suggests that the stabilization of N2terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments. A berrant folding (misfolding) and aggregation of amyloid proteins are pathological hallmarks of a large class of neurodegenerative diseases, including Alzheimer’s disease (AD) (reviewed in 1,2 ). In AD, amyloid precursor proteins are sequentially cleaved by b2secretase and c2secretase, producing the amyloid b2protein (Ab) 3 . Ab peptides contain several alloforms with varying sequences of amino acids, dependent on the cleavage sites of c2secretase. While Ab40 is the most abundant species among these alloforms, Ab42 is the most amyloidogenic species, and also appears to be the most toxic 4–6 . It was shown that the two extra C2terminal residues of Ab42 dramatically change the peptide’s oligomerization properties compared to Ab40 7,8 . Ab42 prefers to form a pentamer/hexamer structure (paranucleus) that can nucleate fibril formation 7 . By contrast, Ab40 reaches rapid equilibrium with coexisting monomers, dimers, trimers, and tetramers 7 . According to computer simulations, Ab42 has a turn2like fold at the C2terminus that involves Met352Gly38 (referred to as a C2turn), suggesting that the more structured C2terminus of Ab42 generates greater intramolecular contacts than Ab40 9 . The hypothesis that the C2terminal residues play an important role in the Ab aggregation process was recently tested by 10 , in which a set of Ab peptides with defined alterations in the C2terminus were studied. Specifically, it was shown that VPV substitutions (Gly33Val2Val36Pro2Gly38Val) stabilized the b2hairpin structure by reducing the flexibility of the peptide backbone and by strengthening hydrophobic interactions between the putative b2strands. A similar VPV motif in an Ab40 mutant changed the oligomerization pattern to one similar to Ab42, resulting in increased toxicity of the oligomers compared with the toxicity of wild type (WT) Ab40 10 . pP substitutions (Val36/D2Pro)2(Gly37/L2Pro) destabilized the putative C2turn structure and thereby made [pP]Ab42 ‘‘Ab402like’’ 10 . We have developed a single2molecule atomic force microscopy (SMFS) approach enabling us to probe and characterize the interactions of the peptides and proteins in transient misfolded conformations 11–15 . In this approach, the protein is end2immobilized on the AFM tip and substrate, allowing us to measure the interprotein interactions at the single2molecule level. The dynamics force spectroscopy (DFS) approach has been quite informative with respect to the lifetimes of transiently formed misfolded dimers (reviewed in 16 ). These studies, applied to Ab40 12 , Ab42 14 , a2synuclein 13 and Sup35 15 , revealed that misfolded dimers have an unexpectedly high stability. Their lifetimes exceeded the characteristic lifetimes of monomers by orders of magnitudes, suggesting SCIENTIFIC REPORTS | 3 : 2880 | DOI: 10.1038/srep02880

Nanoprobing of misfolding and interactions of amyloid β 42 protein

The assembly of amyloid β (Aβ) proteins into nanostructures is currently considered a major pathway of Alzheimer’s disease development, but the molecular mechanisms of this self-assembly process remains unclear. Recently, we showed that single-molecule AFM force spectroscopy (SMFS) is capable of probing the dynamics and interaction between Aβ40 peptides, and these studies allowed us to shed new light on transiently existing Aβ40 misfolding states. In this study, we applied the same SMFS approach to characterize the misfolding of Aβ42 peptide, the most toxic Aβ alloform. The quantitative analysis of SMFS data demonstrated that Aβ interaction leads to the formation of dimers with a lifetime in the range of a second. Interaction via C-terminal segments prevailed at pH 7, but interaction within the peptide center prevailed at acidic pH levels. The difference in the misfolding properties for Aβ40 and Aβ42 peptides and the mechanisms of amyloid nanoassembly are discussed. From the Clinical EditorDespite decades of intense research, Alzheimer's disease still remains incurable. This novel study focuses on the assembly of amyloid β proteins into nanostructures, which is a key mechanism in Alzheimer's disease development. Single molecule atomic force spectroscopy is utilized to shed light on the molecular mechanisms of this self-assembly process.

α-Synuclein Misfolding Assessed with Single Molecule AFM Force Spectroscopy: Effect of Pathogenic Mutations

Misfolding and subsequent aggregation of alpha-synuclein (α-Syn) protein are critically involved in the development of several neurodegenerative diseases, including Parkinson's disease (PD). Three familial single point mutations, A30P, E46K, and A53T, correlate with early onset PD; however, the molecular mechanism of the effects of these mutations on the structural properties of α-Syn and its propensity to misfold remains unclear. Here, we address this issue utilizing a single molecule AFM force spectroscopy approach in which structural details of dimers formed by all four variants of α-Syn are characterized. Analysis of the force spectroscopy data reflecting contour length distribution for α-Syn dimer dissociation suggests that multiple segments are involved in the assembly of the dimer. The interactions are not limited to the central nonamyloid-beta component (NAC) of the protein but rather expand beyond this segment. All three mutations alter the protein's folding and interaction patterns affecting interactions far beyond their immediate locations. Implementation of these findings to our understanding of α-Syn aggregation pathways is discussed.

Single Molecule AFM Force Spectroscopy Analysis of Alpha-Synuclein Misfolding

Protein misfolding is a transient state during self-assembly into aggregates defining the molecular mechanism of the development of Alzheimer's, Parkinson's and other neurodegenerative diseases. Misfolding and aggregation of alpha-synuclein (α-Syn) is tightly linked to the development of Parkinson's disease. Here we applied single molecule AFM force spectroscopy (SMFS) to probe transient misfolded states of α-Syn measuring pair-wise interactions between individual α-Syn molecules at conditions that induce conformational transitions associated with enhanced aggregation. In the SMFS approach we probed the interactions between α-Syn covalently attached to the AFM probe and substrate by the C-terminal cysteine. We show that at conditions close to physiological, addition of spermidine results in dramatic increase of the protein's propensity to misfold. Additionally, using SMFS we detected and characterized misfolded dimers of α-Syn, the simplest aggregated form of α-Syn. Our results demonstrate that more than one segment within the protein molecule is responsible for the initial association of α-Syn into dimers and potentially into higher-order oligomers and fibrils. This finding suggests that even the first step of α-Syn self-assembly (dimerization) possesses a certain degree of heterogeneity. We hypothesize that these different misfolded conformations can lead to different types of oligomers and define the aggregation pathway. The marked differences in the misfolding patterns between WT α-Syn and single point mutants might be responsible for the higher propensity of the mutants to aggregate and cause early-onset PD.The work was supported by grants to YLL from National Institutes of Health (1P01GM091743-01A1 and 1 R01 GM096039-01A1), U.S. Department of Energy Grant DE-FG02-08ER64579, National Science Foundation (EPS - 1004094) and the Nebraska Research Initiative.

Nanoprobing of the Effect of Cu2+ Cations on Misfolding, Interaction and Aggregation of Amyloid Beta Peptide

Misfolding and aggregation of the amyloid β-protein (Aβ) are hallmarks of Alzheimer's disease. Both processes are dependent on the environmental conditions, including the presence of divalent cations, such as Cu2+. Cu2+ cations regulate early stages of Aβ aggregation, but the molecular mechanism of Cu2+ regulation is unknown. In this study we applied single molecule AFM force spectroscopy to elucidate the role of Cu2+ cations on interpeptide interactions. By immobilizing one of two interacting Aβ42 molecules on a mica surface and tethering the counterpart molecule onto the tip, we were able to probe the interpeptide interactions in the presence and absence of Cu2+ cations at pH 7.4, 6.8, 6.0, 5.0, and 4.0. The results show that the presence of Cu2+ cations change the pattern of Aβ interactions for pH values between pH 7.4 and pH 5.0. Under these conditions, Cu2+ cations induce Aβ42 peptide structural changes resulting in N-termini interactions within the dimers. Cu2+ cations also stabilize the dimers. No effects of Cu2+ cations on Aβ-Aβ interactions were observed at pH4.0, suggesting that peptide protonation changes the peptide-cation interaction. The effect of Cu2+ cations on later stages of Aβ aggregation was studied by AFM topographic images. The results demonstrate that substoichiometric Cu2+ cations accelerate the formation of fibrils at pH 7.4 and 5.0, whereas no effect of Cu2+ cations was observed at pH 4.0. Taken together, the combined AFM force spectroscopy and imaging analyses demonstrate that Cu2+ cations promote both the initial and the elongation stages of Aβ aggregation, but protein protonation diminishes the effect of Cu2+.