Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C-terminal alpha helix, modified by a variety of environmental factors as studied by C-13-NMR
S. Yamaguchi et al., Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C-terminal alpha helix, modified by a variety of environmental factors as studied by C-13-NMR, EUR J BIOCH, 268(8), 2001, pp. 2218-2228
We have examined the C-13-NMR spectra of [3-C-13] Ala-labeled bacteriorhodo
psin and its mutants by varying a variety of environmental or intrinsic fac
tors such as ionic strength, temperature, pH, truncation of the C-terminal
a helix, and site-directed mutation at cytoplasmic loops, in order to gain
insight into a plausible surface structure arising from the C-terminal alph
a helix and loops. It is found that the surface structure can be characteri
zed as a complex stabilized by salt bridges or metal-mediated linkages amon
g charged side chains. The surface complex in bacteriorhodopsin is most pro
nounced under the conditions of 10 mM NaCl at neutral pH but is destabilize
d to yield relaxed states when environmental factors are changed to high io
nic strength, low pH and higher temperature. These two states were readily
distinguished by associated spectral changes, including suppressed (cross p
olarization-magic angle spinning NMR) or displaced (upfield) C-13 signals f
rom the C-terminal alpha helix, or modified spectral features in the loop r
egion. It is also noteworthy that such spectral changes, when going from th
e complexed to relaxed states, occur either when the C-terminal alpha helix
is deleted or site-directed mutations were introduced at a cytoplasmic loo
p. These observations clearly emphasize that organization of the cytoplasmi
c surface complex is important in the stabilization of the three-dimensiona
l structure at ambient temperature, and subsequently plays an essential rol
e in biological functions.