Terahertz spectroscopy of bacteriorhodopsin and rhodopsin: similarities and differences

Abstract

We studied the low-frequency terahertz spectroscopy of two photoactive protein systems, rhodopsin and bacteriorhodopsin, as a means to characterize collective low-frequency motions in helical transmembrane proteins. From this work, we found that the nature of the vibrational motions activated by terahertz radiation is surprisingly similar between these two structurally similar proteins. Specifically, at the lowest frequencies probed, the cytoplasmic loop regions of the proteins are highly active; and at the higher terahertz frequencies studied, the extracellular loop regions of the protein systems become vibrationally activated. In the case of bacteriorhodopsin, the calculated terahertz spectra are compared with the experimental terahertz signature. This work illustrates the importance of terahertz spectroscopy to identify vibrational degrees of freedom which correlate to known conformational changes in these proteins.

FIGURE 1

(left) The energy-minimized structure of bacteriorhodopsin and (right) the energy-minimized structure of rhodopsin. The coloring in each figure is as follows, bacteriorhodopsin: Helices A (red), B (yellow), C (light green), D–E (dark green), and F (blue); rhodopsin: Helix I (orange), II (yellow), III (light green), IV–V (dark green), VI (light blue), and VII–VIII (dark blue). The retinal was included in all calculations but is not illustrated in this figure.

FIGURE 2

(a) (Left panel) Overlay of the crystal structure in blue and the energy-minimized structure of bacteriorhodopsin in red and (right panel) overlay of the crystal structure (blue) and energy-minimized structure of rhodopsin (red). The retinal is not illustrated in the figures but is maintained in the calculations. (b) Overlay of the transition state of rhodopsin (red) with the fully minimized structure of rhodopsin (blue).

FIGURE 3

Theoretical terahertz spectra of wild-type bacteriorhodopsin. A Lorentzian representation of the spectra is given as a solid black line. Underneath this is a stick representation of each normal mode calculated, with those highlighted in bold graphically illustrated in Fig. 8.

FIGURE 4

Theoretical terahertz spectra of the D96N mutation of bacteriorhodopsin. A Lorentzian representation of the spectra is given as a solid black line. Underneath this is a stick representation of each normal mode calculated, with those highlighted in bold graphically illustrated in Fig. 8.

FIGURE 5

Theoretical terahertz spectra of rhodopsin. A Lorentzian representation of the spectra is given as a solid black line. Underneath this is a stick representation of each normal mode calculated, with those highlighted in bold graphically illustrated in Fig. 8.

FIGURE 6

Overlay of the three theoretical terahertz spectra of rhodopsin (solid), bacteriorhodopsin (long dash), and the D96D mutant of bacteriorhodopsin (short dash).

FIGURE 7

Overlap of the bacteriorhodopsin experimental spectra (open diamonds) with the predicted terahertz spectra from Fig. 3.

FIGURE 8

Vector representation of selected normal mode motions for the three proteins. The color scheme for the B-factors is as follows: blue illustrates areas of low mobility and red designates areas of high mobility.