28 research outputs found
Mapping the ultrafast vibrational dynamics of all- trans and 13- cis retinal isomerization in Anabaena Sensory Rhodopsin
International audienceDiscrepancies in the isomerization dynamics and quantum yields of the trans and cis retinal protonated Schiff base is a well-known issue in the context of retinal photochemistry. Anabaena Sensory Rhodopsin (ASR) is a microbial retinal protein that comprises a retinal chromophore in two ground state (GS) conformations: all-trans, 15-anti (AT) and 13-cis, 15-syn (13C). In this work, we apply impulsive vibrational spectroscopic techniques (DFWM, pump-DFWM and pump-IVS) to ASR to shed more light on how the structural changes take place in the excited state within the same protein environment. Our findings point to distinct features in the ground state structural conformations as well as to drastically different evolutions in the excited state manifold. The ground state vibrational spectra show stronger Raman activity of the C14-H out-of-plane wag (at about 805 cm-1) for 13C than for AT isomer, which hints at a pre-distortion of the 13C in the ground state. Evolution of the Raman frequency after interaction with actinic pulse shows a blue shift for the C=C stretching and CH3 rocking mode for both isomers. For AT, however, the blue shift is not instantaneous as observed for the 13C isomer, rather it takes more than 200 fs to reach the maximum frequency shift. This frequency blue shift is rationalized by a decrease of the effective conjugation length during the isomerization reaction, which further confirms a slower formation of the twisted state for the AT isomer and corroborates the presence of a barrier in the excited state trajectory previously predicted by quantum chemical calculations
A Color-Determining Amino Acid Residue of Proteorhodopsin
Proteorhodopsin (PR) is a light-driven
proton pump found in marine
bacteria. More than 1000 PRs are classified as blue-absorbing (Ī»<sub>max</sub> ā¼ 490 nm) and green-absorbing (Ī»<sub>max</sub> ā¼ 525 nm) PRs. The color determinant is known to be at position
105, where blue-absorbing and green-absorbing PRs possess Gln and
Leu, respectively. This suggests hydrophobicity at position 105 plays
a key role in color tuning. Here we successfully introduced 19 amino
acid residues into position 105 of green-absorbing PR in the membrane
environment and investigated the absorption properties. High-performance
liquid chromatography analysis shows that the isomeric composition
of the all-<i>trans</i> form is >70% for all mutants,
indicating
little influence of different isomers on color tuning. Absorption
spectra of the wild-type and 19 mutant proteins were well-characterized
by the pH-dependent equilibria of the protonated and deprotonated
counterion (Asp97) of the Schiff base, whereas the Ī»<sub>max</sub> values of these two states and the p<i>K</i><sub>a</sub> value differed significantly among mutants. Although Gln and Leu
are hydrophilic and hydrophobic residues, respectively, the Ī»<sub>max</sub> values of the two states and the p<i>K</i><sub>a</sub> value did not correlate with the hydropathy index of residues.
In contrast, the Ī»<sub>max</sub> and p<i>K</i><sub>a</sub> were correlated with the volume of residues, though Gln and
Leu possess similar volumes. This observation concludes that the Ī»<sub>max</sub> and p<i>K</i><sub>a</sub> of Asp97 are determined
by local and specific interactions in the Schiff base moiety, in which
the volume of the residue at position 105 is more influential than
its hydrophobicity. We suggest that the hydrogen-bonding network in
the Schiff base moiety plays a key role in the Ī»<sub>max</sub> and p<i>K</i><sub>a</sub> of Asp97, and the hydrogen-bonding
network is significantly perturbed by large amino acid residues but
may be preserved by additional water molecule(s) for small amino acid
residues at position 105
FTIR Spectroscopy of a Light-Driven Compatible Sodium Ion-Proton Pumping Rhodopsin at 77 K
<i>Krokinobacter eikastus</i> rhodopsin 2 (KR2) is a
light-driven sodium ion pump that was discovered in marine bacteria.
Although KR2 is able to pump lithium ions similarly, it is converted
into a proton pump in potassium chloride or salts of larger cations.
In this paper, we applied light-induced difference Fourier-transform
infrared (FTIR) spectroscopy to KR2, a compatible sodium ion-proton
pump, at 77 K. The first structural study of the functional cycle
showed that the structure and structural changes in the primary processes
of KR2 are common to all microbial rhodopsins. The red shifted K formation
(KR2<sub>K</sub>) was accompanied by retinal photoisomerization from
an all-trans to a 13-cis form, resulting in a distorted retinal chromophore.
The observed hydrogen out-of-plane vibrations were H/D exchangeable,
indicating that the chromophore distortion by retinal isomerization
is located near the Schiff base region in KR2. This tendency was also
the case for bacteriorhodopsin and halorhodopsin but not the case
for sensory rhodopsin I and II. Therefore, ion pumps such as proton,
chloride, and sodium pumps exhibit local structural perturbations
of retinal at the Schiff base moiety, while photosensors show more
extended structural perturbations of retinal. The retinal Schiff base
of KR2 forms a hydrogen bond that is stronger than in BR. KR2 possesses
more protein-bound water molecules than other microbial rhodopsins
and contains strongly hydrogen-bonded water (OāD stretch at
2333 cm<sup>ā1</sup> in D<sub>2</sub>O). The light-induced
difference FTIR spectra at 77 K were identical between the two states
functioning as light-driven sodium ion and proton pumps, indicating
that the structural changes in the primary processes are identical
between different ion pump functions in KR2. In other words, it is
unknown which ions are transported by molecules when they absorb photons
and photoisomerize. It is likely that the relaxation processes from
the K state lead to an alternative function, namely a sodium ion pump
or proton pump, depending on the environment
A Chimera Na+-Pump Rhodopsin as an Effective Optogenetic Silencer.
With the progress of optogenetics, the activities of genetically identified neurons can be optically silenced to determine whether the neurons in question are necessary for the network performance of the behavioral expression. This logical induction is expected to be improved by the application of the Na+ pump rhodopsins (NaRs), which hyperpolarize the membrane potential with negligible influence on the ionic/pH balance. Here, we made several chimeric NaRs between two NaRs, KR2 and IaNaR from Krokinobacter eikastus and Indibacter alkaliphilus, respectively. We found that one of these chimeras, named I1K6NaR, exhibited some improvements in the membrane targeting and photocurrent properties over native NaRs. The I1K6NaR-expressing cortical neurons were stably silenced by green light irradiation for a certain long duration. With its rapid kinetics and voltage dependency, the photoactivation of I1K6NaR would specifically counteract the generation of action potentials with less hyperpolarization of the neuronal membrane potential than KR2
Functional characterization of sodium-pumping rhodopsins with different pumping properties.
Sodium pumping rhodopsins (NaRs) are a unique member of the microbial-type I rhodopsin family which actively transport Na+ and H+ depending on ionic condition. In this study, we surveyed 12 different NaRs from various sources of eubacteria for their electrophysiological as well as spectroscopic properties. In mammalian cells several of these NaRs exhibited a Na+ based pump photocurrent and four interesting candidates were chosen for further characterization. Voltage dependent photocurrent amplitudes revealed a membrane potential-sensitive turnover rate, indicating the presence of an electrically-charged intermediate(s) in the photocycle reaction. The NaR from Salinarimonas rosea DSM21201 exhibited a red-shifted absorption spectrum, and slower kinetics compared to the first described sodium pump, KR2. Although the ratio of Na+ to H+ ion transport varied among the NaRs we tested, the NaRs from Flagellimonas sp_DIK and Nonlabens sp_YIK_SED-11 showed significantly higher Na+ selectivity when compared to KR2. All four further investigated NaRs showed a functional expression in dissociated hippocampal neuron culture and hyperpolarizing activity upon light-stimulation. Additionally, all four NaRs allowed optical inhibition of electrically-evoked neuronal spiking. Although efficiency of silencing was 3-5 times lower than silencing with the enhanced version of the proton pump AR3 from Halorubrum sodomense, our data outlines a new approach for hyperpolarization of excitable cells without affecting the intracellular and extracellular proton environment
Ultrafast Photoreaction Dynamics of a Light-Driven Sodium-Ion-Pumping Retinal Protein from Krokinobacter eikastus
Unique Hydrogen Bonds in Membrane Protein Monitored by Whole Mid-IR ATR Spectroscopy in Aqueous Solution
Protein
function is coupled to its structural changes, for which
stimulus-induced difference Fourier-transform infrared (FTIR) spectroscopy
is a powerful method. By optimizing the attenuated total reflection
(ATR)-FTIR analysis on sodium-pumping rhodopsin KR2 in aqueous solution,
we first measured the accurate difference spectra upon sodium binding
in the whole IR region (4000ā1000 cm<sup>ā1</sup>).
The new spectral window allows the analysis of not only the fingerprint
region (1800ā1000 cm<sup>ā1</sup>) but also the hydrogen-bonding
donor region (4000ā1800 cm<sup>ā1</sup>), revealing
an unusually strong hydrogen bond of Tyr located in the sodium binding
site of KR2. Progress in ATR-FTIR difference spectroscopy provides an approach to investigating
stimulus-induced structural changes of membrane proteins under physiological
aqueous conditions
Origin of the Reactive and Nonreactive Excited States in the Primary Reaction of Rhodopsins: pH Dependence of Femtosecond Absorption of Light-Driven Sodium Ion Pump Rhodopsin KR2
KR2 is the first light-driven Na<sup>+</sup>-pumping rhodopsin
discovered. It was reported that the photoexcitation of KR2 generates
multiple S<sub>1</sub> states, i.e., āreactiveā and
ānonreactiveā S<sub>1</sub> states at physiological
pH, but their origin remained unclear. In this study, we examined
the S<sub>1</sub> state dynamics of KR2 using femtosecond time-resolved
absorption spectroscopy at different pHā²s in the range from
4 to 11. It was found that the reactive S<sub>1</sub> state is predominantly
formed at pH >9, but its population drastically decreases with
decreasing
pH while the population of the nonreactive S<sub>1</sub> state(s)
increases. The pH dependence of the relative population of the reactive
S<sub>1</sub> state correlates very well with the pH titration curve
of Asp116, which is the counterion of the protonated retinal Schiff
base (PRSB) in KR2. This strongly indicates that the deprotonation/protonation
of Asp116 is directly related to the generation of the multiple S<sub>1</sub> states in KR2. The quantitative analysis of the time-resolved
absorption data led us to conclude that the reactive and nonreactive
S<sub>1</sub> states of KR2 originate from KR2 proteins having a hydrogen
bond between Asp116 and PRSB or not, respectively. In other words,
it is the ground-state inhomogeneity that is the origin of the coexistence
of the reactive and nonreactive S<sub>1</sub> states in KR2. So far,
the generation of multiple S<sub>1</sub> states having a different
photoreactivity of rhodopsins has been mainly explained with the branching
of the relaxation pathway in the FranckāCondon region in the
S<sub>1</sub> state. The present study shows that the structural inhomogeneity
in the ground state, in particular that of the hydrogen-bond network,
is the more plausible origin of the reactive and nonreactive S<sub>1</sub> states which have been widely observed for various rhodopsins
Ultrafast Photoreaction Dynamics of a Light-Driven Sodium-Ion-Pumping Retinal Protein from <i>Krokinobacter eikastus</i> Revealed by Femtosecond Time-Resolved Absorption Spectroscopy
We
report the first femtosecond time-resolved absorption study
on ultrafast photoreaction dynamics of a recently discovered retinal
protein, KR2, which functions as a light-driven sodium-ion pump. The
obtained data show that the excited-state absorption around 460 nm
and the stimulated emission around 720 nm decay concomitantly with
a time constant of 180 fs. This demonstrates that the deactivation
of the S<sub>1</sub> state of KR2, which involves isomerization of
the retinal chromophore, takes place three times faster than that
of bacteriorhodopsin. In accordance with this rapid electronic relaxation,
the photoproduct band assignable to the J intermediate grows up at
ā¼620 nm, indicating that the J intermediate is directly formed
with the S<sub>1</sub> ā S<sub>0</sub> internal conversion.
The photoproduct band subsequently exhibits a ā¼30 nm blue shift
with a 500 fs time constant, corresponding to the conversion to the
K intermediate. On the basis of the femtosecond absorption data obtained,
we discuss the mechanism for the rapid photoreaction of KR2 and its
relevance to the unique function of the sodium-ion pump