Single Molecule Studies of Membrane Proteins on Glass Substrates using Atomic Force Microscopy
Creators
- 1. Johns Hopkins University
- 2. Cal Poly Pomona
- 3. Weill Cornell Medicine
- 4. University of Missouri
Description
1) Method for for integration of high resolution biological AFM with other powerful optical techniques
2) Straight-forward cleaning procedure for treatment of glass for Microscopy and Micromachining applications
3) Molecular high resolution imaging of bacteriorhodopsin and Sec translocase on glass supports
4) Direct observation of protein-protein interactions
5) Atomic Force Microscopy measurements of surface roughness of Glass, Mica, Lipid and comparison of several glass chemical treatments for Microscopy
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Abstract
Since its invention in the mid-1980s, the atomic force microscope (AFM) has become a valuable complementary
tool for studying membrane proteins in near-native environments. Historically, mica is the most common
substrate utilized for biological AFM. Glass being amorphous, transparent, and optically homogeneous has its
own set of advantages over mica and has the potential to broaden the overlap of AFM with techniques that require
high quality non-birefringent optical access. The use of silanized glass as an AFM substrates has been reported as
a means to fine tune surface chemistry. However, such coatings usually require hours of additional preparation
time and can lead to increased surface roughness. In this work, we present a simple technique for preparing
borosilicate glass as a substrate for two membrane protein systems: non-crystalline translocons (SecYEG) of the
general secretary system from E. coli, and bacteriorhodopsin (BR) from H. salinarum. For both these membrane
proteins, quantitative comparisons of the measured protein structures on glass versus mica substrates show
agreement. An additional advantage of glass is that lipid coverage is rapid (< 1 minute) and complete (occupying
the entire surface). A goal is to study the bacterial export system using recently developed precision measurement
techniques such as ultra-stable AFM.
Files
Mid West Single Molecule Workshop 2014 Poster and abstract.pdf
Files
(3.6 MB)
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Additional details
References
- Chada, N., Sigdel, K., Gari, R. et al. Glass is a Viable Substrate for Precision Force Microscopy of Membrane Proteins. Sci Rep 5, 12550 (2015). https://doi.org/10.1038/srep12550
- Chada, N., Sigdel, K., Matin, T., Sanganna Gari, R.R., Mao, C., Randall, L. and King, G., 2013. Glass is a viable substrate for atomic force microscopy of membrane proteins. Bulletin of the American Physical Society, 58. http://meetings.aps.org/Meeting/PSF13/Event/206952
- Chada, N., Sigdel, K.P., Matin, T.R., Gari, R.R.S., Mao, C., Randall, L.L. and King, G.M., 2014. Glass is a viable substrate for atomic force microscopy of membrane proteins. Biophysical Journal, 106(2), p.458a., https://www.cell.com/biophysj/pdf/S0006-3495(13)03853-8.pdf
- Chada, N., Sigdel, K.P., Reddy, R., Gari, S., Matin, T.R., Mao, C., Marsh, B., Randall, L.L. and King, G.M., 2015. Glass: A Multi-Platform Specimen Supporting Substrate for Precision Single Molecule Studies of Membrane Proteins. Biophysical Journal, 108(2), p.170a., https://www.cell.com/biophysj/pdf/S0006-3495(14)02145-6.pdf
- N. Chada, Watching biological nanomotors at work: insights from single-molecule studies, (2017), Thesis, University of Missouri-Columbia, Missouri, USA, Link: , https://mospace.umsystem.edu/xmlui/bitstream/handle/10355/63612/research.pdf.pdf?sequence=1&isAllowed=y
- Chada , Nagaraju . Growth and Characterization of Transition Metal Oxides for Chemical Sensor Applications: Setting up Initiated Hot Wire Chemical Vapor Deposition System. (2010).