Ultrastable atomic force microscopy: Improved force and positional stability

Churnside, Allison B.; Perkins, Thomas T.

doi:10.1016/j.febslet.2014.04.033

Cited by 29 publications

(25 citation statements)

References 104 publications

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“…Second, these cantilevers are inherently stiffer than long cantilevers, leading to increased force noise (δ F = k δ x ) due to low-frequency positional noise (δ x ) in the optical detection systems. 31 , 35 , 36 Finally, such tiny cantilevers are difficult to detect on commercially available AFMs, decreasing usability and throughput. We therefore sought to optimize SMFS with 1-μs temporal resolution by developing soft but ultrashort cantilevers along with the instrumentational improvements to detect them on a commercial AFM.…”

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confidence: 99%

Optimizing 1-μs-Resolution Single-Molecule Force Spectroscopy on a Commercial Atomic Force Microscope

et al. 2015

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Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is widely used to mechanically measure the folding and unfolding of proteins. However, the temporal resolution of a standard commercial cantilever is 50–1000 μs, masking rapid transitions and short-lived intermediates. Recently, SMFS with 0.7-μs temporal resolution was achieved using an ultrashort (L = 9 μm) cantilever on a custom-built, high-speed AFM. By micromachining such cantilevers with a focused ion beam, we optimized them for SMFS rather than tapping-mode imaging. To enhance usability and throughput, we detected the modified cantilevers on a commercial AFM retrofitted with a detection laser system featuring a 3-μm circular spot size. Moreover, individual cantilevers were reused over multiple days. The improved capabilities of the modified cantilevers for SMFS were showcased by unfolding a polyprotein, a popular biophysical assay. Specifically, these cantilevers maintained a 1-μs response time while eliminating cantilever ringing (Q ≅ 0.5). We therefore expect such cantilevers, along with the instrumentational improvements to detect them on a commercial AFM, to accelerate high-precision AFM-based SMFS studies.

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confidence: 99%

Optimizing 1-μs-Resolution Single-Molecule Force Spectroscopy on a Commercial Atomic Force Microscope

et al. 2015

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“…[4,8,9,11,[13][14][15][16][17] Thehigh spatial resolution and fast dynamic response of AFM make it especially appealing for investigating protein dynamics.H owever,t he relatively poor long-term stability of AFM in force [18] makes it challenging to monitor protein folding-unfolding in real time.Although the development of lock-in detection made it possible to observe protein folding, [19] force drift remains the limiting factor for using AFM in protein folding studies. [20][21][22] Recent progress has led to significant improvements in the long-term stability of AFM, allowing for the folding of small proteins to be directly observed in real time and the full characterization of the folding-unfolding energy landscape. [5,19,[22][23][24][25][26] In such experiments,p rotein folding-unfolding at low stretching forces occur under conditions close to equilibrium, giving distinct (un)folding events accompanied with protein shortening (or lengthening).…”

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Direct Observation of the Reversible Two‐State Unfolding and Refolding of an α/β Protein by Single‐Molecule Atomic Force Microscopy

et al. 2015

Angew Chem Int Ed

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Directly observing protein folding in real time using atomic force microscopy (AFM) is challenging. Here the use of AFM to directly monitor the folding of an α/β protein, NuG2, by using low-drift AFM cantilevers is demonstrated. At slow pulling speeds (<50 nm s(-1)), the refolding of NuG2 can be clearly observed. Lowering the pulling speed reduces the difference between the unfolding and refolding forces, bringing the non-equilibrium unfolding-refolding reactions towards equilibrium. At very low pulling speeds (ca. 2 nm s(-1)), unfolding and refolding were observed to occur in near equilibrium. Based on the Crooks fluctuation theorem, we then measured the equilibrium free energy change between folded and unfolded states of NuG2. The improved long-term stability of AFM achieved using gold-free cantilevers allows folding-unfolding reactions of α/β proteins to be directly monitored near equilibrium, opening the avenue towards probing the folding reactions of other mechanically important α/β and all-β elastomeric proteins.

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“…Furthermore, recent technical improvements reducing drift and increasing tip-sample stability to as small as 100 pm have the potential of bringing AFM to single-molecule unfolding experiments that would otherwise employ optical tweezers. 71 Also note that such stability in principle makes previously impossible constant-force experiments feasible. AFM would then hold the advantage that it is more readily automated to scan the surface for viable candidates for stretching; unfold and refold the molecule a predetermined number of times; then break the tether and move on to the next.…”

Section: Afmmentioning

confidence: 99%

−1 Programmed Ribosomal Frameshifting as a Force-Dependent Process

Visscher

2016

Progress in Molecular Biology and Translational Science

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-1 Programmed ribosomal frameshifting is a translational recoding event in which ribosomes slip backward along messenger RNA presumably due to increased tension disrupting the codon-anticodon interaction at the ribosome's coding site. Single-molecule physical methods and recent experiments characterizing the physical properties of mRNA's slippery sequence as well as the mechanical stability of downstream mRNA structure motifs that give rise to frameshifting are discussed. Progress in technology, experimental assays, and data analysis methods hold promise for accurate physical modeling and quantitative understanding of -1 programmed ribosomal frameshifting.

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Ultrastable atomic force microscopy: Improved force and positional stability

Cited by 29 publications

References 104 publications

Optimizing 1-μs-Resolution Single-Molecule Force Spectroscopy on a Commercial Atomic Force Microscope

Optimizing 1-μs-Resolution Single-Molecule Force Spectroscopy on a Commercial Atomic Force Microscope

Direct Observation of the Reversible Two‐State Unfolding and Refolding of an α/β Protein by Single‐Molecule Atomic Force Microscopy

−1 Programmed Ribosomal Frameshifting as a Force-Dependent Process

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