Protein dynamics of a light-driven Na+ pump rhodopsin probed using a tryptophan residue near the retinal chromophore

Abstract

Direct observation of protein structural changes during ion transport in ion pumps provides valuable insights into the mechanism of ion transport. In this study, we examined structural changes in the light-driven sodium ion (Na⁺) pump rhodopsin KR2 on the sub-millisecond time scale, corresponding with the uptake and release of Na⁺. We compared the ion-pumping activities and transient absorption spectra of WT and the W215F mutant, in which the Trp215 residue located near the retinal chromophore on the cytoplasmic side was replaced with a Phe residue. Our findings indicated that atomic contacts between the bulky side chain of Trp215 and the C20 methyl group of the retinal chromophore promote relaxation of the retinal chromophore from the 13-cis to the all-trans form. Since Trp215 is conserved in other ion-pumping rhodopsins, the present results suggest that this residue commonly acts as a mechanical transducer. In addition, we measured time-resolved ultraviolet resonance Raman (UVRR) spectra to show that the environment around Trp215 becomes less hydrophobic at 1 ms after photoirradiation and recovers to the unphotolyzed state with a time constant of around 10 ms. These time scales correspond to Na⁺ uptake and release, suggesting evolution of a transient ion channel at the cytoplasmic side for Na⁺ uptake, consistent with the alternating-access model of ion pumps. The time-resolved UVRR technique has potential for application to other ion-pumping rhodopsins and could provide further insights into the mechanism of ion transport.

Keywords: alternating-access model; microbial rhodopsin; sodium ion pump; time-resolved resonance Raman spectroscopy.

Figure 1

Protein structure of KR2 [PDB ID: 4xto]. An enlarged view shows the retinal chromophore and Trp215.

Figure 2

(A) Ion pump activity of E. coli cells expressing WT (red) and the W215F mutant (blue) of KR2 suspended in 10 mM NaCl, 300 mM Na₂SO₄, and 5% sucrose. Light exposure time is shown by the yellow bars. Light-induced pH changes were monitored without (solid lines) and with (dotted lines) CCCP. (B) Absorption spectra of purified WT (red) and W215F (blue). (C) Visible resonance Raman spectra of WT (red) and W215F (blue) probed by 532 nm laser light. The spectrum of the buffer and the emission background were subtracted.

Figure 3

(A) Transient absorption spectra of WT (top) and W215F (bottom). The delay time corresponding to each color trace is noted at the lower right. (B) Time traces of the difference absorbance at 605 nm of WT (red circles) and 600 nm of W215F (blue squares). The solid lines indicate the fitting curves assuming three time constants.

Figure 4

UVRR spectra of WT (A) and W215F (B). The probe and pump lights were 532 and 233 nm, respectively. The spectral contributions of the buffer and quartz cell were subtracted. The top black traces are the unphotolyzed UVRR spectra measured by only probe light multiplied by a factor of 0.1. The red and blue traces are time-resolved difference spectra obtained by subtracting the spectrum of the unphotolyzed state from the pump-probe spectrum at each delay time.

Figure 5

Temporal relative intensity changes of the W16 and W18 bands observed in the UVRR spectra of WT (A) and W215F (B). The solid red lines indicate the fitting curve ranging from 1 to 8 ms, assuming single exponential decay of the O intermediate. The error bars represent standard errors (N=7 and 5 for WT and W215F, respectively).

Figure 6

Superposition of structures of the retinal chromophore, including the side chain of Lys255 and Trp215 of KR2 in the unphotolyzed state (gold, PDB ID: 6tk6) and at 1 ms (pink, PDB ID: 6tk2). The C20 methyl group of retinal and N_ε1 and C_α of the tryptophan residue are indicated as balls.