Can Dissipative Properties of Single Molecules Be Extracted from a Force Spectroscopy Experiment?

Benedetti, Fabrizio; Gazizova, Yulia; Kulik, Andrzej; Marszałek, Piotr E.; Klinov, Dmitry V.; Dietler, Giovanni; Sekatskii, S. K.

doi:10.1016/j.bpj.2016.08.018

Cited by 13 publications

(43 citation statements)

References 41 publications

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“…With advent of single molecule techniques, such as AFM and optical tweezers, it has been possible to measure internal friction. Recently, doubts have been raised over suitability of small amplitude AFM to detect internal friction in the unfolded protein domains [28]. We clearly show here that dissipation resulting from internal friction can be directly probed with AFM.…”

Section: Discussionmentioning

confidence: 57%

“…While setting up the equation of motion, the cantilever assumed to be a point-mass attached to a massless spring of stiffness k c , and the viscous damping coefficient of γ c . This assumption may work in ultra high vacuum environments [42] and ambient conditions [43], but may lead to incorrect estimation of force in liquid environment [28].…”

Section: Data Analysis Methodsmentioning

confidence: 99%

“…This approximation works well in ambient as well as ultra high vacuum conditions, but fails in situations where it is oscillated in liquid environments. Recently, Benedetti et al have showed that consideration of geometric details of the cantilever is crucial for estimating the dissipation of single molecules [28]. Their improved analysis of the data suggested that molecular dissipation (∼ 10 −7 kg/s) is much lower than the detection limit of AFM.…”

Section: Introductionmentioning

confidence: 99%

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Direct Measurement of Dissipation in a Single Protein using Small Amplitude Atomic Force Microscopy

Rajput

Talele

Ahlawat

et al. 2018

Preprint

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In Krammer's theory, stiffness and dissipation coefficient of a protein determine the rate of their conformational change. Using atomic force microscope, it is possible to measure viscoelasticity of a single protein, wherein it's dissipative and elastic nature is directly and independently measured. Such measurements are performed, either by measuring the thermal fluctuations of the protein held under a constant force, or by providing small modulations to the protein by dithering the cantilever and measuring its response. In small amplitude approximation, where dither amplitude is comparable to persistence length of polymers, it is possible to measure the protein's viscoelastic response accurately. We measured dissipation in I27 at extremely low pulling speeds (∼ 50 nm/s) and low dither frequencies (∼ 100 Hz). At these experimental parameters the dissipation is found to be ∼ 10 −5 kg/s, well above the detection limit of conventional AFM and upper limit predicted by Benedetti et al. Our stiffness data clearly reveals unfolding intermediate of titin's individual immunoglobulin units. The intermediate is elongation of folded domains by ∼ 8Å, wherein two hydrogen bonds are broken between beta sheets. It was possible to measure this elongation in our experiments. The directly measured internal friction of unfolded polymer chain shows a scaling with tension on the chain. The measurements show that it is possible to measure internal friction in single molecules unambiguously using small amplitude AFM. It suggests that systematic experiments to unravel the relation between directly measured internal friction and folding rates of proteins are possible. INTRODUCTIONIn last few decades, a number of experimental techniques have been developed to observe and manipulate matter at atomistic and molecular scales [1][2][3][4][5][6][7][8][9][10]. These developments have a lasting impact on the field of molecular biology as well as on molecular nanotechnology. Single macromolecules such as proteins, flexible polymers and polysaccharides are stretched and coil-to-stretched or folded-to-unfolded transitions under the application of external force are routinely observed. The central goal of these experiments is to mimic the molecular response under natural situations such as ligand-receptor binding, allosteric signalling and the stress induced conformational changes. They measure kinetics of unfolding of a protein and binding affinities of ligands to receptors.Along with optical tweezers [3, 9-12], Atomic Force Microscopy has acquired a unique position in this quest due to its unprecedented spatial resolution in physiological conditions [5][6][7][13][14][15][16][17]. In typical AFM experiment, mica or Au substrate is sparsely coated with the biological macromolecule and is placed in the liquid environment. A sharp probe attached to a cantilever spring is then brought close to a molecule. The protein is allowed to attach to it from either C or N terminus through nonspecific binding. The bending in the cantilever beam is measur...

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Section: Discussionmentioning

confidence: 57%

Section: Data Analysis Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Direct Measurement of Dissipation in a Single Protein using Small Amplitude Atomic Force Microscopy

Rajput

Talele

Ahlawat

et al. 2018

Preprint

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“…Single molecules | viscoelasticity | Atomic Force Microscope | T he viscoelasticity of single proteins and other biologically relevant macromolecules is essential to understand how they function in single molecule limit. Atomic Force Microscopy is used to measure viscoelasticity of single macromolecules and other nano-scale systems owing to its unprecedented spatial resolution in physiological conditions (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). In typical AFM experiment, mica or Au substrate is sparsely coated with the biological macromolecule and is placed in the liquid cell.…”

mentioning

confidence: 99%

“…The cantilever bending provides the amount of force applied on the protein as it is slowly stretched. The amplitude and phase may provide the viscoelastic response of the molecule at different extensions (7,10,11).…”

mentioning

confidence: 99%

Viscoelasticity of single macromolecules using Atomic Force Microscopy

Patil¹,

Rajput²,

deopa³

et al. 2020

Preprint

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We measured viscoelasticity of single protein molecules using two types of Atomic Force Microscopes (AFM) which employ different detection schemes to measure the cantilever response. We used a commonly available deflection detection scheme in commercial AFMs which measures cantilever bending and a fibre-interferometer based home-built AFM which measures cantilever displacement. For both methods, the dissipation coefficient of a single macromolecule is immeasurably low. The upper bound on the dissipation coefficient is 5 × 10 −7 kg/s whereas the entropic stiffness of single unfolded domains of protein measured using both methods is in the range of 10 mN/m. We show that in a conventional deflection detection measurement, the phase of bending signal can be a primary source of artefacts in the dissipation estimates. It is recognized that the measurement of cantilever displacement, which does not have phase lag due to hydrodynamics of the cantilever, is better suited for ensuring artefact-free measurement of viscoelasticty compared to the measurement of the cantilever bending. We confirmed that the dissipation coefficient in single macromolecules is below the detection limit of AFM by measuring dissipation in water layers confined between the tip and the substrate using similar experimental parameters. Further, we experimentally determined the limits in which the simple point-mass approximation of the cantilever works in off-resonance operation.Single molecules | viscoelasticity | Atomic Force Microscope |

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Effect of adsorbate viscoelasticity on dynamical responses of laminated microcantilever resonators

Lyu

Zhang

et al. 2020

Composite Structures

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Can Dissipative Properties of Single Molecules Be Extracted from a Force Spectroscopy Experiment?

Cited by 13 publications

References 41 publications

Direct Measurement of Dissipation in a Single Protein using Small Amplitude Atomic Force Microscopy

Direct Measurement of Dissipation in a Single Protein using Small Amplitude Atomic Force Microscopy

Viscoelasticity of single macromolecules using Atomic Force Microscopy

Effect of adsorbate viscoelasticity on dynamical responses of laminated microcantilever resonators

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