Comparing AFM cantilever stiffness measured using the thermal vibration and the improved thermal vibration methods with that of an SI traceable method based on MEMS

Brand, Uwe; Gao, Shuang; Engl, W.; Sulzbach, T.; Ståhl, S.; Milles, Lukas F.; Нестеров, В. В.; Li, Zhi

doi:10.1088/1361-6501/28/3/034010

Cited by 18 publications

(12 citation statements)

References 31 publications

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“…The tiny differences observed in between cells and between single-cell and single-molecule experiments are likely due to variability of cells and random orientation of the Fg on the AFM tip. But, we cannot exclude that (i) the uncertainty on the calibration of the cantilever spring constant, and (ii) AFM datasets obtained with different cantilevers (thus of different spring constants, see Supplementary Tables 1 and 2 ) 28 also play a role in such small variability. We believe this strong interaction force originates from a DLL binding mechanism because: (i) biochemical analyses demonstrated that SpsD binds Fg through such mechanism, (ii) the high force, much stronger than that of typical protein–receptor interaction (from one to few hundreds of pN), is in the range of values recently reported for adhesins engaged in a DLL interaction 18 , 19 .…”

Section: Resultsmentioning

confidence: 99%

Force-clamp spectroscopy identifies a catch bond mechanism in a Gram-positive pathogen

et al. 2020

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Physical forces have profound effects on cellular behavior, physiology, and disease. Perhaps the most intruiguing and fascinating example is the formation of catch-bonds that strengthen cellular adhesion under shear stresses. Today mannose-binding by the Escherichia coli FimH adhesin remains one of the rare microbial catch-bond thoroughly characterized at the molecular level. Here we provide a quantitative demonstration of a catch-bond in living Gram-positive pathogens using force-clamp spectroscopy. We show that the dock, lock, and latch interaction between staphylococcal surface protein SpsD and fibrinogen is strong, and exhibits an unusual catch-slip transition. The bond lifetime first grows with force, but ultimately decreases to behave as a slip bond beyond a critical force (~1 nN) that is orders of magnitude higher than for previously investigated complexes. This catch-bond, never reported for a staphylococcal adhesin, provides the pathogen with a mechanism to tightly control its adhesive function during colonization and infection.

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Section: Resultsmentioning

confidence: 99%

Force-clamp spectroscopy identifies a catch bond mechanism in a Gram-positive pathogen

et al. 2020

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“…As calibration errors of up to 15% come with cantilever-based force measurements, 29 the precision and therefore the comparability of individual SMFS experiments is limited by the accuracy of the calibration of the cantilever spring constant. This limitation can be circumvented by presenting a set of molecules in a covalently linked microarray format on a single glass slide in predetermined positions.…”

Section: Resultsmentioning

confidence: 99%

Combining in Vitro and in Silico Single-Molecule Force Spectroscopy to Characterize and Tune Cellulosomal Scaffoldin Mechanics

et al. 2017

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Cellulosomes are poly-protein machineries that efficiently degrade cellulosic material. Crucial to their function are scaffolds consisting of highly homologous cohesin domains, which serve a dual role by coordinating a multiplicity of enzymes as well as anchoring the microbe to its substrate. Here we combined two approaches to elucidate the mechanical properties of the main scaffold ScaA of Acetivibrio cellulolyticus. A newly developed parallelized onepot in vitro transcription-translation and protein pulldown protocol enabled high-throughput atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) measurements of all cohesins from ScaA with a single cantilever, thus promising improved relative force comparability. Albeit very similar in sequence, the hanging cohesins showed considerably lower unfolding forces than the bridging cohesins, which are subjected to force when the microbe is anchored to its substrate. Additionally, all-atom steered molecular dynamics (SMD) simulations on homology models offered insight into the process of cohesin unfolding under force. Based on the differences among the individual force propagation pathways and their associated correlation communities, we designed mutants to tune the mechanical stability of the weakest hanging cohesin. The proposed mutants were tested in a second high-throughput AFM SMFS experiment revealing that in one case a single alanine to glycine point mutation suffices to more than double the mechanical stability. In summary, we have successfully characterized the force induced unfolding behaviour of all cohesins from the scaffoldin ScaA, as well as revealed how small changes in sequence can have large effects on force resilience in cohesin domains. Our strategy provides an efficient way to test and improve the mechanical integrity of protein domains in general.

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“…By using a single cantilever through all conditions, calibration errors of up to ~15% that normally result from individual AFM-based SMFS measurements can be circumvented 20 . We therefore obtain comparable absolute force data of all probed cohesins both with and without the dockerin bound.…”

Section: Resultsmentioning

confidence: 99%

Ligand Binding Stabilizes Cellulosomal Cohesins as Revealed by AFM-based Single-Molecule Force Spectroscopy

Verdorfer

Gaub

2018

Sci Rep

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The cohesin-dockerin receptor-ligand family is the key element in the formation of multi-enzyme lignocellulose-digesting extracellular complexes called cellulosomes. Changes in a receptor protein upon binding of a ligand - commonly referred to as allostery - are not just essential for signalling, but may also alter the overall mechanical stability of a protein receptor. Here, we measured the change in mechanical stability of a library of cohesin receptor domains upon binding of their dockerin ligands in a multiplexed atomic force microscopy-based single-molecule force spectroscopy experiment. A parallelized, cell-free protein expression and immobilization protocol enables rapid mechanical phenotyping of an entire library of constructs with a single cantilever and thus ensures high throughput and precision. Our results show that dockerin binding increases the mechanical stability of every probed cohesin independently of its original folding strength. Furthermore, our results indicate that certain cohesins undergo a transition from a multitude of different folds or unfolding pathways to a single stable fold upon binding their ligand.

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Comparing AFM cantilever stiffness measured using the thermal vibration and the improved thermal vibration methods with that of an SI traceable method based on MEMS

Cited by 18 publications

References 31 publications

Force-clamp spectroscopy identifies a catch bond mechanism in a Gram-positive pathogen

Force-clamp spectroscopy identifies a catch bond mechanism in a Gram-positive pathogen

Combining in Vitro and in Silico Single-Molecule Force Spectroscopy to Characterize and Tune Cellulosomal Scaffoldin Mechanics

Ligand Binding Stabilizes Cellulosomal Cohesins as Revealed by AFM-based Single-Molecule Force Spectroscopy

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