“…30 Strong anticipated applications of the presented tip localization method with subsequent nano-structure alignment are single-molecule cut and paste arrangements of enzymatic circuits like replication machineries or cellulosomes and furthermore force spectroscopy in zero-mode waveguides. 31,16 With high throughput efficiencies comparable to those of standard force spectroscopy, the presented approach could become state-of-the-art in the biomolecular research of forceactivated biomolecules 32 by direct observation of enzymatic substrate turnover. 33 Since zero-mode waveguides are applicable to most biophysical assays, 23,[34][35][36] this optomechanical methodology could give insight into many mechanoenzymatic processes obscured so far, especially on the single-molecule level.…”
Since the atomic force microscope (AFM) has evolved into a general purpose platform for mechanical experiments at the nanoscale, the need for a simple and generally applicable localization of the AFM cantilever in the reference frame of an optical microscope has grown. Molecular manipulations like in single molecule cut and paste or force spectroscopy as well as tip mediated nanolithography are prominent examples for the broad variety of applications implemented to date. In contrast to the different kinds of superresolution microscopy where fluorescence is used to localize the emitter, we, here, employ the absorbance of the tip to localize its position in transmission microscopy. We show that in a low aperture illumination, the tip causes a significant reduction of the intensity in the image plane of the microscope objective when it is closer than a few hundred nm. By independently varying the z-position of the sample slide, we could verify that this diffraction limited image of the tip is not caused by a near field effect but is rather caused by the absorbance of the transmitted light in the low apex needle-like tip. We localized the centroid position of this tip image with a precision of better than 6 nm and used it in a feedback loop to position the tip into nano-apertures of 110 nm radius. Single-molecule force spectroscopy traces on the unfolding of individual green fluorescent proteins within the nano-apertures showed that their center positions were repeatedly approached with very high fidelity leaving the specific handle chemistry on the tip's surface unimpaired.
“…30 Strong anticipated applications of the presented tip localization method with subsequent nano-structure alignment are single-molecule cut and paste arrangements of enzymatic circuits like replication machineries or cellulosomes and furthermore force spectroscopy in zero-mode waveguides. 31,16 With high throughput efficiencies comparable to those of standard force spectroscopy, the presented approach could become state-of-the-art in the biomolecular research of forceactivated biomolecules 32 by direct observation of enzymatic substrate turnover. 33 Since zero-mode waveguides are applicable to most biophysical assays, 23,[34][35][36] this optomechanical methodology could give insight into many mechanoenzymatic processes obscured so far, especially on the single-molecule level.…”
Since the atomic force microscope (AFM) has evolved into a general purpose platform for mechanical experiments at the nanoscale, the need for a simple and generally applicable localization of the AFM cantilever in the reference frame of an optical microscope has grown. Molecular manipulations like in single molecule cut and paste or force spectroscopy as well as tip mediated nanolithography are prominent examples for the broad variety of applications implemented to date. In contrast to the different kinds of superresolution microscopy where fluorescence is used to localize the emitter, we, here, employ the absorbance of the tip to localize its position in transmission microscopy. We show that in a low aperture illumination, the tip causes a significant reduction of the intensity in the image plane of the microscope objective when it is closer than a few hundred nm. By independently varying the z-position of the sample slide, we could verify that this diffraction limited image of the tip is not caused by a near field effect but is rather caused by the absorbance of the transmitted light in the low apex needle-like tip. We localized the centroid position of this tip image with a precision of better than 6 nm and used it in a feedback loop to position the tip into nano-apertures of 110 nm radius. Single-molecule force spectroscopy traces on the unfolding of individual green fluorescent proteins within the nano-apertures showed that their center positions were repeatedly approached with very high fidelity leaving the specific handle chemistry on the tip's surface unimpaired.
“…Direct measurements of force-induced substrate binding and activity, that is, RLC phosphorylation, will then lead to full comprehension of this alternative activation path for smMLCK. To this end, the powerful combination of single-molecule force spectroscopy and fluorescence spectroscopy in nanoapertures can provide the basis for in vitro force-activation assays (Heucke et al, 2013), to complement the presented findings that RLC substrate binds smMLCK under force in the absence of Ca 2+ /CaM.10.7554/eLife.26473.030Figure 5.Structural interpretation of the stabilized S state upon RLC interaction.Mechnical stress forces the construct into a conformational state S equivalent to the state reached by Ca 2+ /CaM binding. By release of the pseudosubstrate sequence the conformational state is capable of RLC binding which is detected by a significant stabilization of the S state.…”
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.DOI:
http://dx.doi.org/10.7554/eLife.26473.001
“…The combined use of ZMW and AFM has already been shown feasible in proof of concept studies by using the AFM cantilever tip in surface scanning mode in order to align tip and ZMW. [ 13,14 ] Yet, manual control, cantilever degradation, and small datasets have impeded broad applicability. After mechanical manipulation of a force‐activatable kinase only a single possible binding event was reported.…”
Section: Introductionmentioning
confidence: 99%
“…After mechanical manipulation of a force‐activatable kinase only a single possible binding event was reported. [ 13 ]…”
The mechanobiology of receptor–ligand interactions and force‐induced enzymatic turnover can be revealed by simultaneous measurements of force response and fluorescence. Investigations at physiologically relevant high labeled substrate concentrations require total internal reflection fluorescence microscopy or zero mode waveguides (ZMWs), which are difficult to combine with atomic force microscopy (AFM). A fully automatized workflow is established to manipulate single molecules inside ZMWs autonomously with noninvasive cantilever tip localization. A protein model system comprising a receptor–ligand pair of streptavidin blocked with a biotin‐tagged ligand is introduced. The ligand is pulled out of streptavidin by an AFM cantilever leaving the receptor vacant for reoccupation by freely diffusing fluorescently labeled biotin, which can be detected in single‐molecule fluorescence concurrently to study rebinding rates. This work illustrates the potential of the seamless fusion of these two powerful single‐molecule techniques.
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