Characterization of the motion of membrane proteins using high-speed atomic force microscopy

Casuso, Ignacio; Khao, Jonathan; Chami, Mohamed; Paul‐Gilloteaux, Perrine; Husain, Mohamed; Duneau, Jean‐Pierre; Stahlberg, Henning; Sturgis, James N.; Scheuring, Simon

doi:10.1038/nnano.2012.109

Cited by 188 publications

(193 citation statements)

References 31 publications

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“…We note that the membrane-reconstituted proteins adsorbed to the HS-AFM support may experience somewhat asymmetric conditions. However, the solvent cleft between the membrane and mica is >1 nm, because proteins in less dense reconstitutions are diffusing and hence not directly interacting with the mica (22), and the trimerization domain that protrudes ∼1 nm from the membrane further elevates the membrane surface from the mica; hence, this should allow for rapid diffusion of Na + and Asp to the support-facing side of the membrane. Thus, we believe that the concentrations of Na + ions and Asp, as seen by the transporter, are the same on the opposite sides of the membrane.…”

Section: Significancementioning

confidence: 99%

Direct visualization of glutamate transporter elevator mechanism by high-speed AFM

Ruan

Miyagi

Wang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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108

107

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Glutamate transporters are essential for recovery of the neurotransmitter glutamate from the synaptic cleft. Crystal structures in the outward-and inward-facing conformations of a glutamate transporter homolog from archaebacterium Pyrococcus horikoshii, sodium/aspartate symporter Glt Ph , suggested the molecular basis of the transporter cycle. However, dynamic studies of the transport mechanism have been sparse and indirect. Here we present highspeed atomic force microscopy (HS-AFM) observations of membranereconstituted Glt Ph at work. HS-AFM movies provide unprecedented real-space and real-time visualization of the transport dynamics. Our results show transport mediated by large amplitude 1.85-nm "elevator" movements of the transport domains consistent with previous crystallographic and spectroscopic studies. Elevator dynamics occur in the absence and presence of sodium ions and aspartate, but stall in sodium alone, providing a direct visualization of the ion and substrate symport mechanism. We show unambiguously that individual protomers within the trimeric transporter function fully independently.Glt Ph | HS-AFM | transporter | elevator mechanism | dynamics

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Section: Significancementioning

confidence: 99%

Direct visualization of glutamate transporter elevator mechanism by high-speed AFM

Ruan

Miyagi

Wang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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108

107

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“…High-speed AFM (HS-AFM; (16)) allows imaging biomolecules at video rate (17)(18)(19), through miniaturization of piezoelectric elements and the cantilever (20).…”

Section: Improved Computational Abilities Have Allowed Simulations Whmentioning

confidence: 99%

High-Speed Force Spectroscopy Unfolds Titin at the Velocity of Molecular Dynamics Simulations

Rico

González

Casuso

et al. 2013

Science

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Bridging the Titin Gap The muscle protein titin is a molecular spring that has been extensively studied by single-molecule unfolding experiments and by molecular simulation. However, experimental and simulated unfolding could not be compared directly because they differ by orders of magnitude in pulling velocity. Rico et al. (p 741 ) developed high-speed force spectroscopy to pull titin molecules at speeds that reach the lower limits of molecular dynamics simulations. Bridging the gap between simulation and experiment clarified the mechanism of conformational changes in titin.

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“…From this observation, it was revealed that this inter-trimer bR-bR contact engenders both positive and (Ruan et al 2017) Height change in MloK1 cyclic nucleotide-modulated channels (Kowal et al 2014;Rangl et al 2016) Spiral spring formation by ESCRT protein (Chiaruttini et al 2015) Stiffness map of membrane protein moieties estimated from thermal motion (Preiner et al 2015) Diffusion and interaction of outer membrane protein F (OmpF) (Casuso et al 2012) Bacteriorhodopsin responding to light Yamashita et al 2013) ATP-induced height change of Ca 2+ pump (Yokokawa and Takeyasu Moving net-like structure formed by prion covering living bacterial surface (Yamashita et al 2012;Oestreicher et al 2015) Dynamic morphological changes in living cells (Shibata et al 2015) Dynamics of nucleoporins inside nuclear porin complexes of Xenopus laevis oocytes (Sakiyama et al 2016) Responses of bacteria to antimicrobial peptide (Fantner et al 2010) a Personal communications negative cooperative effects in the decay kinetics as the initial bR recovers. Further HS-AFM studies of bR successfully revealed how bR molecules form trimers and why the trimer formation is required for the function of bR (Yamashita et al 2013).…”

Section: Hs-afm Imaging Of Biological Molecules In Actionmentioning

confidence: 99%

Directly watching biomolecules in action by high-speed atomic force microscopy

Ando

2017

Biophys Rev

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Proteins are dynamic in nature and work at the single molecule level. Therefore, directly watching protein molecules in dynamic action at high spatiotemporal resolution must be the most straightforward approach to understanding how they function. To make this observation possible, highspeed atomic force microscopy (HS-AFM) has been developed. Its current performance allows us to film biological molecules at 10-16 frames/s, without disturbing their function. In fact, dynamic structures and processes of various proteins have been successfully visualized, including bacteriorhodopsin responding to light, myosin V walking on actin filaments, and even intrinsically disordered proteins undergoing order/disorder transitions. The molecular movies have provided insights that could not have been reached in other ways. Moreover, the cantilever tip can be used to manipulate molecules during successive imaging. This capability allows us to observe changes in molecules resulting from dissection or perturbation. This mode of imaging has been successfully applied to myosin V, peroxiredoxin and doublet microtubules, leading to new discoveries. Since HS-AFM can be combined with other techniques, such as super-resolution optical microscopy and optical tweezers, the usefulness of HS-AFM will be further expanded in the near future.

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Characterization of the motion of membrane proteins using high-speed atomic force microscopy

Cited by 188 publications

References 31 publications

Direct visualization of glutamate transporter elevator mechanism by high-speed AFM

Direct visualization of glutamate transporter elevator mechanism by high-speed AFM

High-Speed Force Spectroscopy Unfolds Titin at the Velocity of Molecular Dynamics Simulations

Directly watching biomolecules in action by high-speed atomic force microscopy

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