Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C‐terminal α helix, modified by a variety of environmental factors as studied by ¹³C‐NMR

Abstract

We have examined the 13 C-NMR spectra of [3-13 C] Alalabeled bacteriorhodopsin and its mutants by varying a variety of environmental or intrinsic factors 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 a helix and loops. It is found that the surface structure can be characterized as a complex stabilized by salt bridges or metal-mediated linkages among charged side chains. The surface complex in bacteriorhodopsin is most pronounced under the conditions of 10 mm NaCl at neutral pH but is destabilized to yield relaxed states when environmental factors are changed to high ionic strength, low pH and higher temperature. These two states were readily distinguished by associated spectral changes, including suppressed (cross polarization-magic angle spinning NMR) or displaced (upfield) 13 C signals from the C-terminal a helix, or modified spectral features in the loop region. It is also noteworthy that such spectral changes, when going from the complexed to relaxed states, occur either when the C-terminal a helix is deleted or sitedirected mutations were introduced at a cytoplasmic loop. These observations clearly emphasize that organization of the cytoplasmic surface complex is important in the stabilization of the three-dimensional structure at ambient temperature, and subsequently plays an essential role in biological functions.

Dynamic structure of pharaonis phoborhodopsin (sensory rhodopsin II) and complex with a cognate truncated transducer as revealed by site‐directed 13C solid‐state NMR

“…Such a change is most distinct for the C-terminal K-helix (resonated at 15.9 ppm) protruding from the membrane surface (Fig. 3B) [12^14, 26,27].…”

Site‐directed 13C solid‐state NMR studies on membrane proteins: strategy and goals toward revealing conformation and dynamics as illustrated for bacteriorhodopsin labeled with [1‐13C]amino acid residues

“…In addition, it should be taken into account that the 13 C NMR signals of fully 13 C-labeled proteins, such as [1,2,3-13 C]Ala-labeled bacteriorhodopsin (bR), were substantially broadened owing to the accelerated transverse relaxation rate coupled with inherent fluctuation motions of membrane proteins promoted by increased numbers of relaxation pathways through 13 C- 13 C dipolar interactions and scalar couplings. 2 Accordingly, it is anticipated that the application of the recently developed multi-dimensional 13 C solid-state NMR 3,4 approach will not always be an effective remedy to overcome this problem as far as intact whole membrane proteins integrated in lipid bilayer are concerned.…”

Change in local dynamics of bacteriorhodopsin with retinal isomerization under pressure as studied by fast magic angle spinning NMR

“…At 10 and 12 kHz, the changes in the spectral pattern may be due to increased temperature, because similar NMR spectra have been observed at elevated temperature. 34 After the fast-spinning experiments, the 13 C CP-MAS NMR spectrum of pressure-adapted bR at a MAS-spinning frequency of 4 kHz remained mostly unchanged compared with that of dark-adapted bR at 4kHz. The only change observed was the reduction in the peak intensity at 16.6-16.8 p.p.m.…”

“…The NMR signal of the C-terminal a-helix of Ala228 and 233 at 15.9 p.p.m., which was used as a temperature indicator, slightly shifts to lower values with increasing MAS frequency in the same manner as the temperature variation. 34 After fast sample spinning, the signal of the C-terminal a-helix returned to its initial position at 15.9 p.p.m. at 4 kHz (Figure 1f).…”