Irreversible conformational change of bacterio-opsin induced by binding ofretinal during its reconstitution to bacteriorhodopsin, as studied by C-13NMR
S. Yamaguchi et al., Irreversible conformational change of bacterio-opsin induced by binding ofretinal during its reconstitution to bacteriorhodopsin, as studied by C-13NMR, J BIOCHEM, 127(5), 2000, pp. 861-869
We compared C-13 NMR spectra of [3-C-13]Ala- and [1-C-13]Val-labeled bacter
io-opsin (bO), produced either by bleaching bR with hydroxylamine or from a
retinal-deficient strain, with those of bacteriorhodopsin (bR), in order t
o gain insight into the conformational changes of the protein backbone that
lead to correct folding after retinal is added to bO. The observed C-13 NM
R spectrum of bO produced by bleaching is not greatly different from that o
f bR, except for the presence of suppressed or decreased peak-intensities.
From careful evaluation of the intensity differences between cross polariza
tion magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinni
ng (DD-MAS) spectra, it appears that the reduced peak-intensities arise fro
m reduced efficiency of cross polarization or interference of internal moti
ons with proton decoupling frequencies. In particular, the E-F and F-G loop
s and some transmembrane helices of the bleached bO have acquired-internal
motions whose frequencies interfere with proton decoupling frequencies. In
contrast, the protein backbone of the bO from the retinal-negative cells is
incompletely folded. Although it contains mainly alpha-helices, its very b
road C-13 NMR signals indicate that its tertiary structure is different fro
m bR. Importantly, this changed structure is identical in form to that of b
leached bO from wild-type bR after it was regenerated with retinal in vitro
, and bleached with hydroxylamine. We conclude that the binding of retinal.
is essential for the correct folding of bR after it is inserted in vitro i
nto the lipid bilayer, and the final folded state does not revert to the pa
rtially folded form upon removal of the retinal.