Single-Unit Imaging of Membrane Protein-Embedded Nanodiscs from Two Oriented Sides by High-Speed Atomic Force Microscopy

Abstract

Graphical AbstractHighlights d Membrane protein-embedded nanodiscs were used for highspeed (HS)-AFM observation d Nanodiscs in an end-up and side-on orientation were observed by HS-AFM in real time d HS-AFM combined with nanodiscs is a powerful tool for membrane protein dynamics SUMMARY Membrane proteins play important roles in various cellular functions. To analyze membrane proteins, nanodisc technology using membrane scaffold proteins allows single membrane protein units to be embedded into the lipid bilayer disc without detergents. Recent advancements in high-speed atomic force microscopy (HS-AFM) have enabled us to monitor the real-time dynamics of proteins in solution at the nanometer scale. In this study, we report HS-AFM imaging of membrane proteins reconstituted into nanodiscs using two membrane protein complexes, SecYEG complex and MgtE dimer. The observed images showed single particles of membrane protein-embedded nanodiscs in an end-up orientation whereby the membrane was fixed parallel to the supporting solid surface and in a side-on orientation whereby the membrane plane was vertically fixed to the solid surface, enabling the elucidation of domain fluctuations in membrane proteins. This technique provides a basic method for the high-resolution imaging of single membrane proteins by HS-AFM.

Cryo-EM structure of the MgtE Mg2+channel pore domain in Mg2+-free conditions reveals cytoplasmic pore opening

“…The highly flexible nature of the Mg 2+ -free MgtE cytoplasmic domain in our cryo-EM structure is consistent with previous studies conducted on MgtE by high-speed atomic force microscopy (HS-AFM) (Haruyama et al 2019) and molecular dynamics (MD) simulations (Ishitani et al 2008). In particular, the HS-AFM results directly visualized the continuous shaking motions of the MgtE cytoplasmic domains under Mg 2+ -free conditions.…”

“…In other words, under Mg 2+ -free conditions, the high flexibility of the cytoplasmic domain leads to loss of the interactions between the plug helices and TM domains, unlocking the TM domain conformation from the closed state. Highly flexible domain motions in the MgtE cytoplasmic domain were also observed by high-speed atomic force microscopy (Haruyama et al 2019), in the crystal structure of the Mg 2+ -free cytoplasmic domain structure (Hattori et al 2007), and via MD simulations of MgtE (Ishitani et al 2008) (Fig. 1B and 7B).…”

Three-dimensional reconstruction of individual helical nano-filament structures from atomic force microscopy topographs

“…AFM operating in non-contact mode is capable of reaching atomic resolution on samples of small molecules [7], whereas AFM imaging of biomolecules routinely reaches nanometre resolutions in single high signal-to-noise images, and is able to characterise biological populations at a true single molecule level. This technique has been applied to a range of different bio-molecules, including membrane proteins [8], viral capsids [9] and filamentous biomolecules, such as amyloid fibrils [10], nucleic acids [11,12], and various filaments involved in the cytoskeleton [13].…”

Structural Basis of the Sec Translocon and YidC Revealed Through X-ray Crystallography

“…Moreover, time-dependent structural analyses are also required to further the current understanding of protein transport. Single-molecule analysis helps resolve the underlying mechanism 83,84 , and high-speed atomic force microscopic observations of one unit may provide an overall view of structural changes occurring during protein translocation in real time 85 . Numerous interesting questions regarding a comprehensive understanding of protein transport remain to be answered.…”