Purchase this article with an account.
S. L. Matte, T. M. Laue, R. H. Cote; Analysis of the Structure and Conformational Changes of Photoreceptor PDE6 Using Sedimentation Velocity. Invest. Ophthalmol. Vis. Sci. 2007;48(13):607.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Little is known about the solution structure and conformational properties of PDE6, the central effector enzyme of phototransduction. Using analytical ultracentrifugation with fluorescence detected sedimentation (AU-FDS), picomolar concentrations of fluorescently labeled proteins can be characterized without interference by UV-absorbing compounds. We examined the solution structure of PDE6 subunits and conformational changes induced by Pγ and/or cGMP binding to Pαß.
Purified bovine PDE6 subunits were labeled with 5-iodoacetamidofluorescein (5-IAF) and re-purified to remove unbound dye. Experiments were performed in an Aviv AU-FDS at 50,000 RPM at 20°C. Sedimentation coefficients were determined using Sedfit. Green Fluorescent Protein (2.6 s) served as an internal standard. The Stokes radius (RS) and prolate axial ratio (a/b) were calculated (Sednterp) using the composition MW and the partial specific volume (assuming solvation = 0.4 g-H2O/g-protein).
The sedimentation coefficient of either Pγ/5-IAF or unlabeled Pγ is 1.0 ± 0.1s, consistent with Pγ being asymmetric (RS = 2.22x10-7cm; a/b = 6.6) and natively unfolded. Purified Pαß catalytic dimer (lacking Pγ) sediments at 7.5 ± 0.2 s, consistent with a prolate ellipsoid solution structure (RS= 6.13x10-7cm; a/b = 6.9). While EM particle analysis suggests Pαß to be only a mildly elongated particle, AU-FDS study of the hydrodynamic properties suggest the structure is more highly asymmetric. When a 2-fold excess of Pγ is added back to reform the PDE6 holoenzyme (αßγ2), the sedimentation coefficient increases to 8.0 ± 0.2 s (RS = 6.30x10-7 cm; a/b = 6.8), consistent with an increase in size, not asymmetry. Comparison of PDE6 sedimentation properties with or without cGMP bound to the regulatory GAF domains fail to show significant changes in sedimentation properties upon ligand binding.
Unbound Pγ is likely to exist primarily as a natively unfolded protein. The PDE6 catalytic dimer in solution is highly asymmetric and no major change in asymmetry is detected upon Pγ or cGMP binding to Pαß. This suggests that allosteric changes in PDE6 do not alter its overall three-dimensional structure.
This PDF is available to Subscribers Only