Single molecule studies of membrane proteins on glass substrates using atomic force microscopy Nagaraju Chada1, Krishna P. Sigdel1, Tina R. Matin1, Raghavendar Reddy Sanganna Gari1, Chunfeng Mao2, Linda L. Randall2, and Gavin M. King1,2 1Department of Physics and Astronomy, 2Department of Biochemistry, University of Missouri, Columbia, Missouri, USA 65211 Abstract Since its invention in the mid-1980s, the atomic force microscope1 (AFM) has become an invaluable 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 use the AFM into fields that require high quality non-birefringent optical access. The use of silanized glass as 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 work2, we present a simple technique for preparing borosilicate glass as a substrate for two membrane 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 (< 10 minutes) 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. Why use glass as a substrate?  Typical AFM substrates mica and HOPG exhibit birefringence, complicating optical paths  Many well established biological and biochemical assays like FRAP, FRET, Fluorescence Microscopy, FIONA and TIRF require transparent and optically homogeneous substrates  Recently developed techniques like US-AFM4, 3D AFM and polarization anisotropy methods require transparent substrates and non-birefringence due to polarization based detection  Amorphous and optically homogeneous substrates like glass enable coupling AFM with advanced imaging techniques2 Heights of SecYEG Potential Applications Raw glass vs. KOH treated glass References Acknowledgements This work was supported by the National Science Foundation (CAREER Award #:1054832), the Burroughs Wellcome Fund (Career Award at the Scientific Interface) and the MU Research Board. We thank the JILA Scientific Communications Office for artwork. High Resolution Images of SecYEG Bacteriorhodopsin on Glass and Mica Evaluating Different Cleaning Treatments Sec-translocase  >30% of proteins are transported from the site of synthesis into or through a membrane5  In E. coli, the Sec system orchestrates the translocation of polypeptides across membrane before they acquire stable tertiary structure and SecYEG provides the path way5,6  SecA and SecB act as chaperones to maintain newly synthesized polypeptides in a state compatible with transport6  Numerous questions remain regarding the mechanistic details of translocation  Novel AFM modalities could measure molecular “fly fishing”6  Map trajectories of protein domains in 3D  Dynamic Loops of SecYEG, linking transmembrane helices 6-7 and 8-9, play a vital role in capturing SecA and in translocation  Probing translocation of precursor through Sec- translocase 1. Binnig, G.; Quate, C. F.; Gerber, Ch. (1986). "Atomic Force Microscope". Physical Review Letters. 56 (9): 930–933 2. 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 3. Chada, Nagaraju. "Growth and Characterization of Transition Metal Oxides for Chemical Sensor Applications: Setting up Initiated Hot Wire Chemical Vapor Deposition System."