Abstract
Though ubiquitous in optical microscopy, glass has long been overlooked as a specimen supporting surface for high resolution atomic force microscopy (AFM) investigations due to its roughness. Using bacteriorhodopsin from Halobacterium salinarum and the translocon SecYEG from Escherichia coli, we demonstrate that faithful images of 2D crystalline and non-crystalline membrane proteins in lipid bilayers can be obtained on microscope cover glass following a straight-forward cleaning procedure. Direct comparison between AFM data obtained on glass and on mica substrates show no major differences in image fidelity. Repeated association of the ATPase SecA with the cytoplasmic protrusion of SecYEG demonstrates that the translocon remains competent for binding after tens of minutes of continuous AFM imaging. This opens the door for precision long-timescale investigations of the active translocase in near-native conditions and, more generally, for integration of high resolution biological AFM with many powerful optical techniques that require non-birefringent substrates.
Highlights
Atomic force microscopy (AFM) has emerged as an important tool for macromolecular characterization in biological settings and is well suited for studying membrane proteins, which are challenging to address using traditional techniques[1,2,3]
In this work we sought to couple the benefits of glass substrates with high resolution biological atomic force microscopy (AFM)
As supplied by the manufacturer, borosilicate glass cover slips are rough on the molecular scale (Fig. 1a), exhibiting an average rms roughness of 19 ± 9.6 Å. This limits their direct application in high resolution AFM
Summary
Nagaraju Chada[1], Krishna P. Though ubiquitous in optical microscopy, glass has long been overlooked as a specimen supporting surface for high resolution atomic force microscopy (AFM) investigations due to its roughness. Repeated association of the ATPase SecA with the cytoplasmic protrusion of SecYEG demonstrates that the translocon remains competent for binding after tens of minutes of continuous AFM imaging This opens the door for precision long-timescale investigations of the active translocase in near-native conditions and, more generally, for integration of high resolution biological AFM with many powerful optical techniques that require non-birefringent substrates. Propagation through birefringent material alters the polarization state and bifurcates the propagation direction of light in a manner which varies with material thickness This makes it challenging to utilize freshly cleaved mica surfaces in modern optical systems, many of which employ highly focused and polarized laser beams passing through the specimen plane. We suggest more generally that glass-supported lipid bilayers may be an effective mimic of the situation in vivo wherein numerous punctate contacts are made with membrane, for example, by cytoskeletal elements[30]
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