Abstract
We have used electron cryo-microscopy (cryo-EM) of single particles (individual protein complexes) and two-dimensional crystals along with fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) to determine structures and dynamics of complexes formed by the photoreceptor G protein transducin, and its effector enzyme, cGMP phosphodiesterase, PDE6. Cryo-EM studies of complexes tagged with Fab fragments of monoclonal antibodies revealed that the inhibitory gamma subunit of PDE6, PDE6γ, stretches from the catalytic domain of PDE6 where its carboxyl-terminal region binds, to the GAFa domain, where its amino terminus binds, consistent with previous photo-crosslinking studies. FRET reveals that in unbound PDE6γ the amino and carboxyl termini are fairly close to one another. Upon addition of the catalytic subunits of PDE6, there is an initial very fast (near diffusion limit) binding to the catalytic domain, followed by a very slow (minutes) stretching out and binding of the amino terminal region to the distant GAFa domain. FRAP measurement of diffusion in living rod cells of transgenic Xenopus laevis revealed free diffusion of transducin along the long axis of cell, and the presence in disk membranes of cholesterol-dependent lipid microdomains, into which transducin complexes segregate upon activation.
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