AFM(T) + FLUORESCENCE MEASUREMENTS ON SINGLE MOLECULES

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DESCRIPTION (provided by applicant): Our long-term objectives are to increase molecular-level understanding of select health and disease processes. We seek to achieve this with a new instrument capable of single-molecule fluorescence measurements of angular (polarization), translational, or conformational motions collected before, during, or after AFM-imaging and manipulation of individual biomolecules and their complexes. AFM is the only instrument that can image wet, native samples with nanometer resolution, and it has also emerged as a key technology for manipulation-type studies of single molecules and complexes, including studies of enzyme activity under compressive confinement and protein folding-unfolding under tension begun by Gaub and Fernandez. The Discher lab was the first to demonstrate strong temperature effects on stability of single proteins during forced extension of proteins by AFM(Temp) and more recently showed that protein-protein interactions can detectably couple to unfolding but a large number of molecular biophysics questions remain. Fluorescence microscopy has also progressed over the last decade to single molecule detection, and two of the latest innovations applied to myosin motors come from the Goldman lab and collaborators with direct measurement of rotational motions as well as improved spatial resolution of lateral motions to 1 nm. Cooperman & Goldman have begun to apply these methods to ribosomes. The Shuman lab has likewise developed related, optics-based technology for mechanical interrogation of protein-protein interactions and for energy transduction in molecular motors. To more deeply pursue this NIH-funded work and additional projects, including externally submitted projects (5 wks per yr), we propose to make extensive use of a multi-purpose instrument capable of performing single molecule fluorescence before, during, or after imaging and manipulation by AFM(T=20-60 ¿C). With a few accessories, the fluorescence methods will include two-color Polarization-TIRF, single-pair FRET, and nm-scale distance measurements while we use AFM(T) on the same region to either image or simultaneously perform single molecule mechanical tests on complexes and proteins. Key users of the instrument will investigate at least 7 NIH-funded research projects that are relevant to public health including: ribosome function under stress and confinement of fundamental importance, myosin thick filaments under force relevant to cardiomyopathies, mini-dystrophin and spectrin extensibility relevant to biophysical foundations for gene therapy of both muscular dystrophy and anemias, and cell membrane adhesion of integrin-associated proteins relevant to immune system responses.