Control of Ligand-Binding Specificity Using Photocleavable Linkers in AFM Force Spectroscopy.

In recent decades, atomic force microscopy (AFM), in particular the force spectroscopy mode, has become a method of choice to study biomolecular interactions at the single-molecule level. However, grafting procedures as well as determining binding specificity remain challenging. We report here an innovative approach based on a photocleavable group that enables in situ release of the ligands bound to the AFM tip and thus allows direct assessment of the binding specificity. Applicable to a wide variety of molecules, the strategy presented here provides new opportunities to study specific interactions and deliver single molecules with high spatiotemporal resolution in a wide range of applications, including AFM-based cell biology.

Competitive binding assay for biotin and biotin derivatives, based on avidin and biotin-4-fluorescein.

Biotinylated molecules are extensively employed in bioanalytics and biotechnology. The currently available assays for quantification of biotin groups suffer from low sensitivity, low accuracy, or provide highly variable responses for different biotin derivatives. We developed a competitive binding assay in which avidin was pre-blocked to different extents by the biotinylated analyte and a constant amount of biotin-4-fluorescein (B4F) was added, resulting in strong quenching of the B4F. The assay was robust and the shape of the titration curve immediately revealed whether the data were reliable or perturbed by steric hindrance in case of large biotin derivatives. These advantages justified well the 10× higher sample consumption (~0.6nmol) compared to single point assays. The assay was applied to a representative set of small biotin derivatives and validated by cross-control with the well-established 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) binding assay. In comparison to the 2,6-ANS binding assay, the lower precision (±10%) was compensated by the 100-fold higher sensitivity and the deviations from the ANS assay were ≤5%. In comparison to the more sensitive biotin group assays, the new assay has the advantage of minimal bias for different biotin derivatives. In case of biotinylated DNA with 30 nucleotides, steric hindrance was found to reduce the accuracy of biotin group determination; this problem was overcome by partial digestion to n≤5 nucleotide residues with a 3'-exonuclease. The newly proposed biotin group assay offers a useful compromise in terms of sensitivity, precision, trueness, and robustness.

AFM-Based Force Spectroscopy Guided by Recognition Imaging: A New Mode for Mapping and Studying Interaction Sites at Low Lateral Density.

Ligand binding to receptors is one of the most important regulatory elements in biology as it is the initiating step in signaling pathways and cascades. Thus, precisely localizing binding sites and measuring interaction forces between cognate receptor-ligand pairs leads to new insights into the molecular recognition involved in these processes. Here we present a detailed protocol about applying a technique, which combines atomic force microscopy (AFM)-based recognition imaging and force spectroscopy for studying the interaction between (membrane) receptors and ligands on the single molecule level. This method allows for the selection of a single receptor molecule reconstituted into a supported lipid membrane at low density, with the subsequent quantification of the receptor-ligand unbinding force. Based on AFM tapping mode, a cantilever tip carrying a ligand molecule is oscillated across a membrane. Topography and recognition images of reconstituted receptors are recorded simultaneously by analyzing the downward and upward parts of the oscillation, respectively. Functional receptor molecules are selected from the recognition image with nanometer resolution before the AFM is switched to the force spectroscopy mode, using positional feedback control. The combined mode allows for dynamic force probing on different pre-selected molecules. This strategy results in higher throughput when compared with force mapping. Applied to two different receptor-ligand pairs, we validated the presented new mode.

Two Ligand Binding Sites in Serotonin Transporter Revealed by Nanopharmacological Force Sensing.

The number of ligand binding sites in neurotransmitter-sodium symporters has been determined by crystal structure analysis and molecular pharmacology with controversial results. Here, we designed molecular tools to measure the interaction forces between the serotonin transporter (SERT) and S-citalopram on the single-molecule level by means of atomic force microscopy. Force spectroscopy allows for the extraction of dynamic information under physiological conditions which is inaccessible via X-ray crystallography. Two populations of distinctly different binding strength between S-citalopram and SERT were demonstrated in Na+-containing buffer. In Li+-containing buffer, SERT showed merely low-force interactions, whereas the vestibular mutant SERT-G402H only displayed the high force population. These observations provide physical evidence for the existence of two different binding sites in SERT when tested under near-physiological conditions.

Communication between N terminus and loop2 tunes Orai activation.

Ca2+ release-activated Ca2+ (CRAC) channels constitute the major Ca2+ entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca2+-selective Orai channel and the Ca2+-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating. Although the extended transmembrane Orai N-terminal region (Orai1 amino acids 73-91; Orai3 amino acids 48-65) is fully conserved in the Orai1 and Orai3 isoforms, Orai3 tolerates larger N-terminal truncations than Orai1 in retaining store-operated activation. In an attempt to uncover the reason for these isoform-specific structural requirements, we analyzed a series of Orai mutants and chimeras. We discovered that it was not the N termini, but the loop2 regions connecting TM2 and TM3 of Orai1 and Orai3 that featured distinct properties, which explained the different, isoform-specific behavior of Orai N-truncation mutants. Atomic force microscopy studies and MD simulations suggested that the remaining N-terminal portion in the non-functional Orai1 N-truncation mutants formed new, inhibitory interactions with the Orai1-loop2 regions, but not with Orai3-loop2. Such a loop2 swap restored activation of the N-truncation Orai1 mutants. To mimic interactions between the N terminus and loop2 in full-length Orai1 channels, we induced close proximity of the N terminus and loop2 via cysteine cross-linking, which actually caused significant inhibition of STIM1-mediated Orai currents. In aggregate, maintenance of Orai activation required not only the conserved N-terminal region but also permissive communication of the Orai N terminus and loop2 in an isoform-specific manner.

Regenerative biosensor for use with biotinylated bait molecules.

Label-free biosensors are ideally suited for the quantitative analysis of specific interactions among biomolecules or of biomolecules with drugs, as well as for quantitation of diagnostic markers in biofluids. In contrast to the label-dependent methods, a new assay for a particular prey molecule can be set up within few minutes by immobilizing the corresponding bait molecule on the sensor surface, using one of the common immobilization procedures. Unfortunately, the extensive application of label-free biosensors is still hampered by the fact that the immobilization of the bait molecule is usually irreversible; for that reason, a new chip (which is expensive) is required for every successful or futile attempt. Here, we present a general method for the switchable immobilization of biotinylated bait molecules on a new desthiobiotin surface, using wild-type streptavidin as a robust bridge between the chip and the biotinylated bait. The immobilization of the bait is very stable, so that many cycles of prey injection and subsequent prey removal can be carried out. For the latter, common reagents like HCl, Na2CO3, glycine buffer, or SDS are employed. When desired, however, streptavidin plus the biotinylated bait can be completely removed by 3min injections of biotin, guanidinium thiocyanate, pepsin, and SDS, which makes it possible to immobilize new biotinylated bait. The number of in situ regeneration cycles is unlimited during the lifetime of the chip (2-3 weeks). One chip can easily be shared by many users with unrelated tasks (as is typical in academics), or used for the fully automated screening of many different interactions (for example in pharmaceutical research). In comparison to other regenerative chips, the new chip surface has much wider applicability and all of its structural and functional parameters have been disclosed.

Stable Europium(III) Complexes with Short Linkers for Site-Specific Labeling of Biomolecules.

In this study, two new terpyridine-based EuIII complexes were synthesized, the structures of which were optimized for luminescence resonance energy-transfer (LRET) experiments. The complexes showed high quantum yields (32 %); a single long lifetime (1.25 ms), which was not influenced by coupling to protein; very high stability in the presence of chelators such as ethylenediamine-N,N,N',N'-tetraacetate and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid; and no interaction with cofactors such as adenosine triphosphate and guanosine triphosphate. A special feature is the short length of the linker between the EuIII ion and the maleimide or hydrazide function, which allows for site-specific coupling of cysteine mutants or unnatural keto amino acids. As a consequence, the new complexes appear particularly suited for accurate distance measurements in biomolecules by LRET.

Detailed Evidence for an Unparalleled Interaction Mode between Calmodulin and Orai Proteins.

Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic α-helix. The N-terminus of Orai proteins contains a conserved CaM-binding segment but the binding mechanism has been only partially characterized. Here, microscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy (AFM) were employed to study the binding equilibria, the kinetics, and the single-molecule interaction forces involved in the binding of CaM to the conserved helical segments of Orai1 and Orai3. The results consistently indicated stepwise binding of two separate target peptides to the two lobes of CaM. An unparalleled high affinity was found when two Orai peptides were dimerized or immobilized at high lateral density, thereby mimicking the close proximity of the N-termini in native Orai oligomers. The analogous experiments with smooth muscle myosin light chain kinase (smMLCK) showed only the expected 1:1 binding, confirming the validity of our methods.

Combined Recognition Imaging and Force Spectroscopy: A New Mode for Mapping and Studying Interaction Sites at Low Lateral Density.

We combined recognition imaging and force spectroscopy to study the interactions between receptors and ligands on the single molecule level. This method allowed the selection of a single receptor molecule reconstituted in a supported lipid membrane at low density, with the subsequent quantification of the receptor-ligand unbinding force. Based on atomic force microscopy (AFM) tapping mode, a cantilever tip carrying a ligand molecule was oscillated across a membrane. Topography and recognition images of reconstituted receptors were recorded simultaneously by analyzing the downward and upward parts of the oscillation, respectively. Functional receptor molecules were selected from the recognition image with nanometer resolution before the AFM was switched to the force spectroscopy mode, using positional feedback control. The combined mode allowed for dynamic force probing on different pre-selected molecules, resulting in higher throughput when compared with force mapping. We applied this method for a quantitative characterization of the binding mechanism between mitochondrial uncoupling protein 1 (UCP1) and its inhibitor adenosine triphosphate (ATP). Moreover the dynamics of force loading was varied to elucidate the binding dynamics and map the interaction energy landscape.

Mutual A domain interactions in the force sensing protein von Willebrand factor.

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets.

Curli mediate bacterial adhesion to fibronectin via tensile multiple bonds.

Many enteric bacteria including pathogenic Escherichia coli and Salmonella strains produce curli fibers that bind to host surfaces, leading to bacterial internalization into host cells. By using a nanomechanical force-sensing approach, we obtained real-time information about the distribution of molecular bonds involved in the adhesion of curliated bacteria to fibronectin. We found that curliated E. coli and fibronectin formed dense quantized and multiple specific bonds with high tensile strength, resulting in tight bacterial binding. Nanomechanical recognition measurements revealed that approximately 10 bonds were disrupted either sequentially or simultaneously under force load. Thus the curli formation of bacterial surfaces leads to multi-bond structural components of fibrous nature, which may explain the strong mechanical binding of curliated bacteria to host cells and unveil the functions of these proteins in bacterial internalization and invasion.

Single molecule force spectroscopy data and BD- and MD simulations on the blood protein von Willebrand factor.

We here give information for a deeper understanding of single molecule force spectroscopy (SMFS) data through the example of the blood protein von Willebrand factor (VWF). It is also shown, how fitting of rupture forces versus loading rate profiles in the molecular dynamics (MD) loading-rate range can be used to demonstrate the qualitative agreement between SMFS and MD simulations. The recently developed model by Bullerjahn, Sturm, and Kroy (BSK) was used for this demonstration. Further, Brownian dynamics (BD) simulations, which can be utilized to estimate the lifetimes of intramolecular VWF interactions under physiological shear, are described. For interpretation and discussion of the methods and data presented here, we would like to directly point the reader to the related research paper, "Mutual A domain interactions in the force sensing protein von Willebrand Factor" (Posch et al., 2016) [1].

Nanopharmacological Force Sensing to Reveal Allosteric Coupling in Transporter Binding Sites.

Controversy regarding the number and function of ligand binding sites in neurotransmitter/sodium symporters arose from conflicting data in crystal structures and molecular pharmacology. Here, we have designed novel tools for atomic force microscopy that directly measure the interaction forces between the serotonin transporter (SERT) and the S- and R-enantiomers of citalopram on the single molecule level. This approach is based on force spectroscopy, which allows for the extraction of dynamic information under physiological conditions thus inaccessible via X-ray crystallography. Two distinct populations of characteristic binding strengths of citalopram to SERT were revealed in Na(+)-containing buffer. In contrast, in Li(+) -containing buffer, SERT showed only low force interactions. Conversely, the vestibular mutant SERT-G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERT when accessed in a physiological context. Competition experiments revealed that these two sites are allosterically coupled and exert reciprocal modulation.

Switchavidin: reversible biotin-avidin-biotin bridges with high affinity and specificity.

Switchavidin is a chicken avidin mutant displaying reversible binding to biotin, an improved binding affinity toward conjugated biotin, and low nonspecific binding due to reduced surface charge. These properties make switchavidin an optimal tool in biosensor applications for the reversible immobilization of biotinylated proteins on biotinylated sensor surfaces. Furthermore, switchavidin opens novel possibilities for patterning, purification, and labeling.

IgGs are made for walking on bacterial and viral surfaces.

Binding of antibodies to their cognate antigens is fundamental for adaptive immunity. Molecular engineering of antibodies for therapeutic and diagnostic purposes emerges to be one of the major technologies in combating many human diseases. Despite its importance, a detailed description of the nanomechanical process of antibody-antigen binding and dissociation on the molecular level is lacking. Here we utilize high-speed atomic force microscopy to examine the dynamics of antibody recognition and uncover a principle; antibodies do not remain stationary on surfaces of regularly spaced epitopes; they rather exhibit 'bipedal' stochastic walking. As monovalent Fab fragments do not move, steric strain is identified as the origin of short-lived bivalent binding. Walking antibodies gather in transient clusters that might serve as docking sites for the complement system and/or phagocytes. Our findings could inspire the rational design of antibodies and multivalent receptors to exploit/inhibit steric strain-induced dynamic effects.

Forces and dynamics of glucose and inhibitor binding to sodium glucose co-transporter SGLT1 studied by single molecule force spectroscopy.

Single molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic force microscopy tips carrying either thioglucose, 2'-aminoethyl ß-d-glucopyranoside, or aminophlorizin. Poly(ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations.

Investigating the binding behaviour of two avidin-based testosterone binders using molecular recognition force spectroscopy.

Molecular recognition force spectroscopy, a biosensing atomic force microscopy technique allows to characterise the dissociation of ligand-receptor complexes at the molecular level. Here, we used molecular recognition force spectroscopy to study the binding capability of recently developed testosterone binders. The two avidin-based proteins called sbAvd-1 and sbAvd-2 are expected to bind both testosterone and biotin but differ in their binding behaviour towards these ligands. To explore the ligand binding and dissociation energy landscape of these proteins, we tethered biotin or testosterone to the atomic force microscopy probe while the testosterone-binding protein was immobilized on the surface. Repeated formation and rupture of the ligand-receptor complex at different pulling velocities allowed determination of the loading rate dependence of the complex-rupturing force. In this way, we obtained the molecular dissociation rate (k(off)) and energy landscape distances (x(ß)) of the four possible complexes: sbAvd-1-biotin, sbAvd-1-testosterone, sbAvd-2-biotin and sbAvd-2-testosterone. It was found that the kinetic off-rates for both proteins and both ligands are similar. In contrast, the x(ß) values, as well as the probability of complex formations, varied considerably. In addition, competitive binding experiments with biotin and testosterone in solution differ significantly for the two testosterone-binding proteins, implying a decreased cross-reactivity of sbAvd-2. Unravelling the binding behaviour of the investigated testosterone-binding proteins is expected to improve their usability for possible sensing applications.

Kinetics of bioconjugate nanoparticle label binding in a sandwich-type immunoassay.

Nanoparticle labels have enhanced the performance of diagnostic, screening, and other measurement applications and hold further promise for more sensitive, precise, and cost-effective assay technologies. Nevertheless, a clear view of the biomolecular interactions on the molecular level is missing. Controlling the ratio of molecular recognition over undesired nonspecific adhesion is the key to improve biosensing with nanoparticles. To improve this ratio with an aim to disallow nonspecific binding, a more detailed perspective into the kinetic differences between the cases is needed. We present the application of two novel methods to determine complex binding kinetics of bioconjugate nanoparticles, interferometry, and force spectroscopy. Force spectroscopy is an atomic force microscopy technique and optical interferometry is a direct method to monitor reaction kinetics in second-hour timescale, both having steadily increasing importance in nanomedicine. The combination is perfectly suited for this purpose, due to the high sensitivity to detect binding events and the ability to investigate biological samples under physiological conditions. We have attached a single biofunctionalized nanoparticle to the outer tip apex and studied the binding behavior of the nanoparticle in a sandwich-type immunoassay using dynamic force spectroscopy in millisecond timescale. Utilization of the two novel methods allowed characterization of binding kinetics in a time range spanning from 50 ms to 4 h. These experiments allowed detection and demonstration of differences between specific and nonspecific binding. Most importantly, nonspecific binding of a nanoparticle was reduced at contact times below 100 ms with the solid-phase surface.

The extended transmembrane Orai1 N-terminal (ETON) region combines binding interface and gate for Orai1 activation by STIM1.

STIM1 and Orai1 represent the two molecular key components of the Ca(2+) release-activated Ca(2+) channels. Their activation involves STIM1 C terminus coupling to both the N terminus and the C terminus of Orai. Here we focused on the extended transmembrane Orai1 N-terminal (ETON, aa73-90) region, conserved among the Orai family forming an elongated helix of TM1 as recently shown by x-ray crystallography. To identify "hot spot" residues in the ETON binding interface for STIM1 interaction, numerous Orai1 constructs with N-terminal truncations or point mutations within the ETON region were generated. N-terminal truncations of the first four residues of the ETON region or beyond completely abolished STIM1-dependent Orai1 function. Loss of Orai1 function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1 interaction. In a complementary approach, we monitored STIM1-Orai interaction via Orai1 V102A by determining restored Ca(2+) selectivity as a consequence of STIM1 coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca(2+) selectivity of Orai1 V102A in the presence of STIM1, demonstrating impairment of STIM1 binding. Hence, the major portion of the ETON region (aa76-90) is essential for STIM1 binding and Orai1 activation. Mutagenesis within the ETON region revealed several hydrophobic and basic hot spot residues that appear to control STIM1 coupling to Orai1 in a concerted manner. Moreover, we identified two basic residues, which protrude into the elongated pore to redound to Orai1 gating. We suggest that several hot spot residues in the ETON region contribute in aggregate to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate.