Mechanical unfolding of a2‐macroglobulin molecules with atomic force microscope

Mitsui, Kiyofumi; Hara, Masahiko; Ikai, Atsushi

doi:10.1016/0014-5793(96)00319-5

Cited by 130 publications

(68 citation statements)

References 17 publications

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“…The sandwiched sample will then be stretched until one or more of the weaker bonds in the system are broken. During the stretching process of the protein molecule, the mechanical response of the sample against the tensile stress produces a downward deflection of the cantilever which holds the probe at its free end (Mitsui et al 1996). The extent of cantilever deflection, d, is detected by a bisected photodiode detector and, by knowing the cantilever force constant, the tensile force inflicted on the sample is calculated as FZKkd.…”

Section: Results (A) Protein Stretching Experimentsmentioning

confidence: 99%

Nanobiomechanics of proteins and biomembrane

Ikai

2008

Phil. Trans. R. Soc. B

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A review of the work done in the Laboratory of Biodynamics of Tokyo Institute of Technology in the last decade has been summarized in this article in relation to the results reported from other laboratories. The emphasis here is the application of nanomechanics based on the force mode of atomic force microscopy (AFM) to proteins and protein-based biological structures. Globular proteins were stretched in various ways to detect the localized rigidity inside of the molecule. When studied by this method, bovine carbonic anhydrase II (BCA II), calmodulin and OspA protein all showed the presence of localized rigid structures inside the molecules. Protein compression experiments were done on BCA II to obtain an estimate of the Young modulus and its change in the process of denaturation. Then, the AFM probe method was turned on to cell membranes and cytoplasmic components. Force curves accompanying the extraction process of membrane proteins from intact cells were analysed in relation to their interaction with the cytoskeletal components. By pushing the AFM probe further into the cytoplasm, mRNAs were recovered from a live cell with minimal damage, and multiplied using PCR technology for their identification. Altogether, the work introduced here forms the basis of nanomechanics of protein and protein-based biostructures and application of the nanomechanical technology to cell biology.

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Section: Results (A) Protein Stretching Experimentsmentioning

confidence: 99%

Nanobiomechanics of proteins and biomembrane

Ikai

2008

Phil. Trans. R. Soc. B

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“…The new experimental techniques have been employed in protein folding studies where mechanical force was used as a denaturation method. [11][12][13] Here, to examine whether the monomeric unfolding intermediate of GroES actually exists during unfolding, we applied this mechanical unfolding method to GroES. A GroES protein whose subunits had been covalently linked was immobilized on mica and extended (unfolded) mechanically in solution using AFM.…”

Section: Introductionmentioning

confidence: 99%

Mechanical unfolding of covalently linked GroES: Evidence of structural subunit intermediates

et al. 2008

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It is difficult to determine the structural stability of the individual subunits or protomers of many proteins in the cell that exist in an oligomeric or complexed state. In this study, we used single-molecule force spectroscopy on seven subunits of covalently linked cochaperonin GroES (ESC7) to evaluate the structural stability of the subunit. A modified form of ESC7 was immobilized on a mica surface. The force-extension profile obtained from the mechanical unfolding of this ESC7 showed a distinctive sawtooth pattern that is typical for multimodular proteins. When analyzed according to the worm-like chain model, the contour lengths calculated from the peaks in the profile suggested that linkedGroES subunits unfold in distinct steps after the oligomeric ring structure of ESC7 is disrupted. The evidence that structured subunits of ESC7 withstand external force to some extent even after the perturbation of the oligomeric ring structure suggests that a stable monomeric intermediate is an important component of the equilibrium unfolding reaction of GroES.Keywords: force spectroscopy; mechanical unfolding intermediates; protein subunit stability; covalently linked oligomeric protein; GroES; cpn10Abbreviations: AFM, atomic force microscopy; CH-ESC7, ESC7 that has two-cysteine residues and six-histidine residues extending from the N terminus and C terminus, respectively; ESC7, engineered form of cochaperonin GroES with covalently linked subunits; Gdn-HCl, guanidine hydrochloride; PBS, phosphate saline buffer.

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“…One of the alternative techniques that have recently been used to study protein unfolding is the force spectroscopy method based on the atomic force microscopy (AFM; Mitsui et al 1996;Zlatanova et al 2000). This novel technique uses an extremely sensitive force sensor of AFM that is able to detect forces in the range of pico-to nanonewtons.…”

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Unfolding mechanics of holo‐ and apocalmodulin studied by the atomic force microscope

Hertadi¹,

Ikai²

2002

Protein Science

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The structural stability of calmodulin (CaM) has been investigated previously by chemical and thermal methods. The calcium-loaded form of CaM has been found to be exceptionally stable, because it can be exposed to temperatures >90°C or to a 9 M urea solution without a marked change in its tertiary structure, and is therefore not experimentally accessible for unfolding studies using conventional analytical methods. In this study, we have developed a system for measuring the force for mechanically unfolding CaM using an atomic force microscope (AFM) by stretching the protein from its N-and C-terminal residues; we have been successful in obtaining force versus extension (F-E) curves for both apo and holo forms of CaM. In our experiment, distinguishable F-E curves have been obtained upon stretching of apoCaM and holoCaM to their full extensions. A very low force observed upon stretching of apoCaM indicated a relatively high flexibility of the apo form. On the contrary, a relatively high unfolding force and the appearance of a characteristic force peak were noted during full stretching of holoCaM. The F-E curve of the latter form of CaM most likely reflects a more rigid and probably more organized conformation of holoCaM than that of apoCaM. These experiments confirmed that the AFM is able to clearly distinguish two functionally distinct forms of CaM in terms of their mechanical properties.

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Mechanical unfolding of a2‐macroglobulin molecules with atomic force microscope

Cited by 130 publications

References 17 publications

Nanobiomechanics of proteins and biomembrane

Nanobiomechanics of proteins and biomembrane

Mechanical unfolding of covalently linked GroES: Evidence of structural subunit intermediates

Unfolding mechanics of holo‐ and apocalmodulin studied by the atomic force microscope

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