IDBR: High throughput single molecule AFM force-spectrometer

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An award has been made to Columbia University to develop the first high throughput single molecule AFM force-spectrometer. Single-molecule force spectroscopy by atomic force microscopy (AFM) is widely used to measure the dynamic of proteins placed under force, of common occurrence in Biology. A growing number of biologists are taking advantage of the detailed information that emerges from force-spectroscopy measurements. However, current force-spectrometers face huge limitations since they are remarkably low-throughput. The aims of the proposal are designed to overcome the limitations of AFM spectrometers and aims to construct a new instrument where throughput is increased by at least two orders of magnitude. Together with the construction of the new instrument, novel technologies for the covalent attachment of the molecules will be developed which will allow for long-term recordings (hours to days) from a single protein molecule placed under force. In order to analyze the large volume of data anticipated, new methods of analysis based on Extended Kalman Filters will be implemented. By increasing at least by a factor of 100 the throughput of force-spectroscopy by AFM, new capabilities will be accessible to a wide community of Biologists interested in protein dynamics, protein mechanics and other disciplines. Currently, there is a growing consensus that mechanical perturbations of proteins are commonplace in vivo. However, it is extremely challenging even to conceive experiments to find small molecules that affect the mechanical properties of a protein, such as mechanical stability. Such compounds would be extraordinarily useful to understand the role of mechanical forces in physiology and disease, and they may even find therapeutic application. Being able to test hundreds of small molecules in a working day will allow the identification of such small molecules. This interdisciplinary proposal will be used to train undergraduate, graduate and postdoctoral students in the art of instrument design, and single molecule recordings. Both undergraduate and graduate students will be actively involved in the design and construction of the high-throughput force spectrometer. In particular, undergraduate students always find interdisciplinary research very attractive and make use of summer research programs to engage in interdisciplinary projects, such as the one described in this proposal. A graduate student and a postdoctoral fellow will work full time in different aspects of the proposal. The proposal includes a mentoring plan for the postdoctoral fellow that will ease his transition to an independent position, increasing his chances to become a successful member of the research community. The dissemination of the results is crucial to the success of the proposal. The ultimate goal of the proposal is to make the new instrumentation available through partnerships with companies, in combination with presentations in conferences and workshops.Funded through the Instrument Development for Biological Research program in the Division of Biological Infrastructure.