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Force-induced remodelling of proteins and their complexes.

Chen Y, Radford SE, Brockwell DJ - Curr. Opin. Struct. Biol. (2015)

Bottom Line: The effects of force on the biophysical properties of biological systems can be large and varied.As these effects are only apparent in the presence of force, studies on the same proteins using traditional ensemble biophysical methods can yield apparently conflicting results.Where appropriate, therefore, force measurements should be integrated with other experimental approaches to understand the physiological context of the system under study.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK; School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.

No MeSH data available.


Experimental set-up and comparison of the key parameters, features and limitations of (left) atomic force microscopy (AFM), (middle) optical tweezers and (right) on-cell patch clamping.
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Figure 1: Experimental set-up and comparison of the key parameters, features and limitations of (left) atomic force microscopy (AFM), (middle) optical tweezers and (right) on-cell patch clamping.

Mentions: To analyse and understand the effects of force at the molecular level it is necessary to manipulate single bio-molecules or perturb their environment. This review discusses three commonly used techniques that achieve this feat: atomic force microscopy (AFM), optical or magnetic tweezers and patch clamping. As summarised in Figure 1 each method has its own optimal force and distance resolutions and experimental limitations. The application of AFM to force measurements (sometimes called force spectroscopy or dynamic force spectroscopy, DFS) has gained widespread use in biology. It is able to measure single molecule stretching and rupture forces directly with subnanometer distance and picoNewton force resolution [3]. This technique is typically used to measure relatively high forces in biological terms (pN — nN) at relatively high extension rates (10–10000 nm s−1). However, both limitations have been addressed by the use of uncoated [4] and nano-engineered cantilevers [5•], pushing the lower force threshold to the subpicoNewton level. Tweezers use either light (laser tweezers/optical traps) or magnetic fields (magnetic tweezers) as the force transducer [6,7]. Laser tweezers have been used to study processive motors, such as myosin [8], kinesin [9] and ClpX [10,11], while magnetic tweezers are applied most often to torque-generating proteins such as those that interact with DNA [12]. Both techniques are now being applied to study mechanical unfolding, with optical trapping technology able to study multiple folding/unfolding cycles of a single protein over many seconds [13]. On-cell patch-clamp is an electrophysiological technique that is especially powerful for the study of single ion channels on intact cells. By attaching a micropipette to the cell membrane, the current created by a single ion channel can be recorded [14]. For force studies, patch clamp offers the opportunity to assess the role of the lipid environment on membrane proteins by changing membrane tension or modulating lipid identity. The utility of these techniques can also be enhanced by combining them with other methods. For example, the combination of force and fluorescence spectroscopy provides a powerful tool to gain information simultaneously on single molecule forces and conformational changes (including complex formation) induced by force [15,16].


Force-induced remodelling of proteins and their complexes.

Chen Y, Radford SE, Brockwell DJ - Curr. Opin. Struct. Biol. (2015)

Experimental set-up and comparison of the key parameters, features and limitations of (left) atomic force microscopy (AFM), (middle) optical tweezers and (right) on-cell patch clamping.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499843&req=5

Figure 1: Experimental set-up and comparison of the key parameters, features and limitations of (left) atomic force microscopy (AFM), (middle) optical tweezers and (right) on-cell patch clamping.
Mentions: To analyse and understand the effects of force at the molecular level it is necessary to manipulate single bio-molecules or perturb their environment. This review discusses three commonly used techniques that achieve this feat: atomic force microscopy (AFM), optical or magnetic tweezers and patch clamping. As summarised in Figure 1 each method has its own optimal force and distance resolutions and experimental limitations. The application of AFM to force measurements (sometimes called force spectroscopy or dynamic force spectroscopy, DFS) has gained widespread use in biology. It is able to measure single molecule stretching and rupture forces directly with subnanometer distance and picoNewton force resolution [3]. This technique is typically used to measure relatively high forces in biological terms (pN — nN) at relatively high extension rates (10–10000 nm s−1). However, both limitations have been addressed by the use of uncoated [4] and nano-engineered cantilevers [5•], pushing the lower force threshold to the subpicoNewton level. Tweezers use either light (laser tweezers/optical traps) or magnetic fields (magnetic tweezers) as the force transducer [6,7]. Laser tweezers have been used to study processive motors, such as myosin [8], kinesin [9] and ClpX [10,11], while magnetic tweezers are applied most often to torque-generating proteins such as those that interact with DNA [12]. Both techniques are now being applied to study mechanical unfolding, with optical trapping technology able to study multiple folding/unfolding cycles of a single protein over many seconds [13]. On-cell patch-clamp is an electrophysiological technique that is especially powerful for the study of single ion channels on intact cells. By attaching a micropipette to the cell membrane, the current created by a single ion channel can be recorded [14]. For force studies, patch clamp offers the opportunity to assess the role of the lipid environment on membrane proteins by changing membrane tension or modulating lipid identity. The utility of these techniques can also be enhanced by combining them with other methods. For example, the combination of force and fluorescence spectroscopy provides a powerful tool to gain information simultaneously on single molecule forces and conformational changes (including complex formation) induced by force [15,16].

Bottom Line: The effects of force on the biophysical properties of biological systems can be large and varied.As these effects are only apparent in the presence of force, studies on the same proteins using traditional ensemble biophysical methods can yield apparently conflicting results.Where appropriate, therefore, force measurements should be integrated with other experimental approaches to understand the physiological context of the system under study.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK; School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.

No MeSH data available.