? Protein deformation, protein folding, and protein interaction with other proteins (e.g., receptor-ligand binding) are all mediated by piconewton or femtonewton level forces. Only one of the existing low force application techniques can be used to impose forces of femtonewton level. Therefore, the overall goal of this proposal is to develop and validate a novel technique of imposing femtonewton level forces. The goal of the R21 phase of this proposal is to conduct a comprehensive numerical analysis and an experimental validation of the proposed technique (EMAT) to assess its feasibility.
The specific aims of the R21 phase are: 1) to conduct an axisymmetric analysis of the EMAT, 2) to conduct a three-dimensional analysis of the EMAT, and 3) to validate the EMAT with ultra-soft springs. The goal of the R33 phase of this proposal is to build a database that can be used by other researchers to calculate forces for various combinations of all the parameters involved, to build a device that can be used to further validate the EMAT experimentally, and to apply it to single receptor-ligand interactions.
The specific aims of the R33 Phase are: 1) to develop a computer program that can be used by others to easily calculate the force imposed with the EMAT, 2) to implement a computer-controlled micropipette manipulation system that can be used to impose femtonewton level forces, 3) to further validate the EMAT with optical tweezers, and 4) to examine the effect of force on the interaction between L-selectin and its ligands. The analyses proposed in the R21 phase will be performed with FIDAP (a finite element analysis software package for computational fluid dynamics) to evaluate the effects of some critical parameters and the alignment between the micropipette and transducer on the calculated force. The validation of the EMAT will be carried out with the micropipette manipulation system, ultra-soft springs, and optical tweezers. Combined with the micropipette aspiration technique, the EMAT will enable us to impose a wide range of forces from a few femtonewtons to about 100 nanonewtons, which is unprecedented. Therefore, the EMAT should have great potential to make an impact in the field of single molecule and single bond biophysics. ? ?
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