Early-stage dynamics of chloride ion–pumping rhodopsin revealed by a femtosecond X-ray laser

Abstract

Chloride ion–pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl− into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation–diffusion process upon light-triggered retinal isomerization.

Unidirectional ion transport mechanism of a light-driven chloride pump revealed using X-ray free electron lasers

“…Meanwhile, Trp99 and Trp201, located around the retinal, were displaced from 1 ps to 100 ps as observed in the NM-R3-I data at Δt=10 μs (32). Therefore, photoisomerization of the retinal may trigger the displacements of Cltoward Thr102 and two tryptophan residues, followed by the movement of Asn98 in helix C to fill in the space left after the Clmoved.…”

“…The NM-R3-I structure at Δ t =10 μs is presumed to be the L(N) state, since the K state appears at an earlier step after light illumination. Interestingly, TR-SFX studies of NM-R3 that measured early-stage dynamics (1-100 ps) showed that Cl - is already translocated to Thr102 upon retinal isomerization at Δ t =100 ps and the C α of Asn98 slightly moved within 1 Å (32), in contrast to the movements in the NM-R3-I structure at Δ t =10 μs. Meanwhile, Trp99 and Trp201, located around the retinal, were displaced from 1 ps to 100 ps as observed in the NM-R3-I data at Δ t =10 μs (32).…”

“…Interestingly, TR-SFX studies of NM-R3 that measured early-stage dynamics (1-100 ps) showed that Cl - is already translocated to Thr102 upon retinal isomerization at Δ t =100 ps and the C α of Asn98 slightly moved within 1 Å (32), in contrast to the movements in the NM-R3-I structure at Δ t =10 μs. Meanwhile, Trp99 and Trp201, located around the retinal, were displaced from 1 ps to 100 ps as observed in the NM-R3-I data at Δ t =10 μs (32). Therefore, photoisomerization of the retinal may trigger the displacements of Cl - toward Thr102 and two tryptophan residues, followed by the movement of Asn98 in helix C to fill in the space left after the Cl - moved.…”

“…Combined with a visible-light pump laser for reaction initiation, TR-SFX has been applied to light-driven ion pumps to observe the structural dynamics during the ion transfer. While TR-SFX has revealed femto-to-millisecond structural dynamics in lightdriven cation pumps, including bR and KR2 (29)(30)(31), TR-SFX studies of anion pumps have been limited to early-stage structures adopted at picoseconds after light illumination (32). In addition, although NM-R3 pumps a chloride ion (Cl -) as a physiological substrate, it can also transport bromide (Br -), iodide (I -) and other anions from the extracellular side to the cytoplasmic side (23).…”

Protein structure, dynamics, and function—a 20th IUPAB Congress symposium

“…With these, he was able to explain the structural transition itself as well as determining the timescale over which it occurs. In a second example, the role of a conformational change within the chloride channel was elegantly used to explain the pumping mechanism across the membrane (Yun et al 2021). However, most biological systems are not light-sensitive, so a rapid mix and inject strategy needs to be employed (Pandey et al 2021).…”