15N MAS NMR studies of cph1 phytochrome: Chromophore dynamics and intramolecular signal transduction.
T. Rohmer, H. Strauss, J. Hughes, H. de Groot, W. Gartner, P. Schmieder, J. Matysik
J Phys Chem B (2006) 110, 20580-20585
Solid-state nuclear magnetic resonance (NMR) is applied for the first time to the photoreceptor phytochrome. The two stable states, Pr and Pfr, of the 59-kDa N-terminal module of the cyanobacterial phytochrome Cph1 from Synechocystis sp. PCC 6803 containing a uniformly 15N-labeled phycocyanobilin cofactor are explored by 15N cross-polarization (CP) magic-angle spinning (MAS) NMR. As recently shown by 15N solution-state NMR using chemical shifts [Strauss, H. M.; Hughes, J.; Schmieder, P. Biochemistry 2005, 44, 8244], all four nitrogens are protonated in both states. CP/MAS NMR provides two additional independent lines of evidence for the protonation of the nitrogens. Apparent loss of mobility during photoactivation, indicated by the decrease of line width, demonstrates strong tension of the entire chromophore in the Pfr state, which is in clear contrast to a more relaxed Pr state. The outer rings (A and D) of the chromophore are significantly affected by the phototransformation, as indicated by both change of chemical shift and line width. On the other hand, on the inner rings (B and C) only minor changes of chemical shifts are detected, providing evidence for a conserved environment during phototransformation. In a mechanical model, the phototransformation is understood in terms of rotations between the A-B and C-D methine bridges, allowing for intramolecular signal transduction to the protein surface by a unit composed of the central rings B and C and its tightly linked protein surroundings during the highly energetic Pfr state.