| Fluorescence spectroscopy provides a wide variety of techniques for studying the conformational changes which proteins undergo as they function. In this thesis I present experiments on two different proteins using a variety of fluorescence techniques.; Fluorescence Resonance Energy Transfer (FRET) is a technique for measuring distances (2–10nm) shorter than the wavelength of light (∼500nm) by exploiting the ability of two fluorescent dyes to transfer energy via interaction of their transition dipole moments with an efficiency that depends on their separation. Lanthanide-based Resonance Energy Transfer (LRET) is a modification of the technique in which a luminescent lanthanide chelate, such as Tb 3+-DTPA-cs124, is used in place of a conventional organic fluorophore.; The first protein I studied is the Shaker potassium channel, a voltage gated ion channel selective for potassium ions. We used LRET to measure distances between identical sites on the four subunits of Shaker and find no evidence for the traditional model of S4 motion, a large translation across the membrane. Rather, our results suggest a model in which S4 undergoes the more subtle motion of a rotation and possibly a tilt.; The other protein I studied is myosin V which walks bipedally along actin filaments taking steps which displace the center of mass 37 nm per step. A controversy over the nature of this movement was settled earlier this year by other members of our laboratory when they showed that myosin V processes in a hand-over-hand manner, with each of the heads alternating in the leading and trailing positions. To do this, they developed a new technique which they called FIONA for Fluorescence Imaging with One Nanometer Accuracy which allowed them to localize individual molecules of myosin V to within 1.5 nut on the surface of a microscope cover slip. I have extended and applied FIONA to a mutant of myosin V with a molecule of the enhanced Green Fluorescent Protein (eGFP) fused to one head of the dieter. I demonstrated that eGFP is suitable to use with FIONA, confirm the hand-over-hand model of myosin procession, and argue that in aggregate, the data on myosin's steps from our lab implies that myosin V adopts an asymmetric, “telemark skier,” configuration between steps. (Abstract shortened by UMI.)... |
