Small and Mechanosensitive Membrane Proteins Studied with DNA-based Tools

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Project Summary I am requesting funds to purchase an Atomic Force Microscope (AFM) to replace an aged and failing AFM that requires frequent major repairs. AFM is a vital method for the work proposed in the parent R35 grant which will provide new DNA-based tools that will overcome several of the challenges associated with MP structure deter- mination by single-molecule cryo EM. An AFM is needed for the development phase of our DNA-lipid nanodiscs, as AFMs produce images of individual particles with a higher resolution, contrast and signal to noise ratio than other imaging methods and no other method provides topographical information. Instant access to this instrument in our laboratory avoids long wait times thus shortening the cycles of iterative improvements of our DNA-based tools. AFM sample preparation is also much faster and requires less material than TEM grid sample preparations while avoiding staining, drying and transfer into a vacuum. In AFM, samples are imaged in native buffers, instead. The new instrument supports imaging with video frame rates which would allow us to study dynamic processes including nanodisc fusion and lipid exchange. Moreover, the accelerated imaging dramatically reduces the time that is required to image a statistically relevant number of particles which is needed for a biophysical quantification of structural parameters and synthesis yields of the DNA rings. Finally, the requested instrument will be the first modern AFM on the campus and I therefore expect interesting collaboration requests that will leverage our expertise in AFM.