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.
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.
The cytokine tumor necrosis factor-alpha (TNF-α) readily forms homotrimers at sub-nM concentrations to promote inflammation. For the treatment of inflammatory diseases with upregulated levels of TNF-α, a number of therapeutic antibodies are currently used as scavengers to reduce the active TNF-α concentration in patients. Despite their clinical success, the mode-of-action of different antibody formats with regard to a stabilization of the trimeric state is not entirely understood. Here, we use a biosensor with dynamic nanolevers to analyze the monomeric and trimeric states of TNF-α together with the binding kinetics of therapeutic biologics. The intrinsic trimer-to-monomer decay rate k = 1.7 × 10−3 s−1 could be measured directly using a microfluidic system, and antibody binding affinities were analyzed in the pM range. Trimer stabilization effects are quantified for Adalimumab, Infliximab, Etanercept, Certolizumab, Golimumab for bivalent and monovalent binding formats. Clear differences in trimer stabilization are observed, which may provide a deeper insight into the mode-of-action of TNF-α scavengers.
We developed an impedance quartz crystal microbalance (QCM) approach with the ability to simultaneously record mass changes and calibrated energy dissipation with high sensitivity using an impedance analyzer. This impedance QCM measures frequency shifts and resistance changes of sensing quartz crystals very stable, accurately, and calibrated, thus yielding quantitative information on mass changes and dissipation. Resistance changes below 0.3 Ω were measured with corresponding dissipation values of 0.01 µU (micro dissipation units). The broadband impedance capabilities allow measurements between 20 Hz and 120 MHz including higher harmonic modes of up to 11th order for a 10 MHz fundamental resonance frequency quartz crystal. We demonstrate the adsorbed mass, calibrated resistance, and quantitative dissipation measurements on two biological systems including the high affinity based avidin-biotin interaction and nano-assemblies of polyelectrolyte layers. The binding affinity of a protein-antibody interaction was determined. The impedance QCM is a versatile and simple method for accurate and calibrated resistance and dissipation measurements with broadband measurement capabilities for higher harmonics measurements.
Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic a-helix. The N-terminus of Orai proteins contains ac onserved CaM-binding segment but the binding mechanismh as been only partially characterized. Here,m icroscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy( AFM) were employed to study the binding equilibria, the kinetics,a nd 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.T he analogous experiments with smooth muscle myosin light chain kinase (smMLCK) showed only the expected 1:1b inding,c onfirming the validity of our methods. Orai1andOrai3areCa2+ channels in the plasma membrane of non-excitable cells which are activated by Ca 2+ depletion of the endoplasmic reticulum (ER);areduction of [Ca 2+ ]inthe ER causes the stromal interaction molecule (STIM) in the ER membrane to oligomerize,w hereupon it binds to Orai and activates its channel function. [1][2][3][4] Much less is known about the Ca 2+ -dependent inactivation (CDI) of Orai which is important in the regulation of the intracellular Ca 2+ level. In anumber of studies,calmodulin (CaM) was found to bind to ahighly conserved N-terminal segment of Orai1/3, and to act as an egative regulator of channel function, [5][6][7] although this was lately questioned.[8] Thec rystal structure of the complex between CaM and the isolated CaM-binding segment of Orai1 (aa69-88) revealed an unusual extended conformation of CaM with only its C-terminal lobe binding one Orai1 peptide.[9] Parallel pulldown and isothermal titration calorimetry (ITC) experiments suggested stepwise binding of two Orai1 69-88 peptides with different affinities,o ne on the Cterminal lobe of CaM (K d = 1.1 mm)a nd one on the Nterminal lobe (K d = 4.6 mm). Motivated by these findings,weaimed at acomprehensive characterization of this interaction mechanism, for the following reasons:( i) the measured K d values (1.1 and 4.6 mm)a re much higher than those usually reported for Ca 2+ -induced CaM binding (typically 10 À7 to 10 À11 m) [10] and were probably influenced by the high calmodulin concentration used in ITC,( ii)the transitions between monovalent and bivalent bond formation have not yet been addressed, and, most prominently,( iii)quantitative information about the kinetics of stepwise association and dissociation is still missing. Here,t hese questions were addressed by complementary in vitro methods.E quilibrium measurements by MST yielded the distinct affinities of both binding steps at equilibrium, SPR provided information about the kinetic rate constants,a nd AFM revealed the interaction forces of monovalent and bivalent binding,aswell as the...
Dynamic methods of biosensing based on electrical actuation of surface-tethered nanolevers require the use of levers whose movement in ionic liquids is well controllable and stable. In particular, mechanical integrity of the nanolevers in a wide range of ionic strengths will enable to meet the chemical conditions of a large variety of applications where the specific binding of biomolecular analytes is analyzed. Herein, we study the electrically induced switching behavior of different rodlike DNA origami nanolevers and compare to the actuation of simply double-stranded DNA nanolevers. Our measurements reveal a significantly stronger response of the DNA origami to switching of electrode potential, leading to a smaller potential change necessary to actuate the origami and subsequently to a long-term stable movement. Dynamic measurements in buffer solutions with different Mg 2+ contents show that the levers do not disintegrate even at very low ion concentrations and constant switching stress and thus provide stable actuation performance. The latter will pave the way for many new applications without largely restricting application-specific environments.
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