Transmembrane ion gradients are generated and maintained by ion-pumping proteins in cells. Light-driven ion-pumping rhodopsins are retinal-containing proteins found in archaea, bacteria, and eukarya. ...Photoisomerization of the retinal chromophore induces structural changes in the protein, allowing the transport of ions in a particular direction. Understanding unidirectional ion transport by ion-pumping rhodopsins is an exciting challenge for biophysical chemistry. Concerted changes in ion-binding affinities of the ion-binding sites in proteins are key to unidirectional ion transport, as is the coupling between the chromophore and the protein moiety to drive the concerted motions regulating ion-binding affinities. The commonality of ion-pumping rhodopsin protein structures and the diversity of their ion-pumping functions suggest universal principles governing ion transport, which would be widely applicable to molecular systems. In this Perspective, I review the insights obtained from previous studies on rhodopsins and discuss future perspectives.
Vibrational energy exchange between various degrees of freedom is critical to barrier-crossing processes in proteins. Hemeproteins are well suited for studying vibrational energy exchange in proteins ...because the heme group is an efficient photothermal converter. The released energy by heme following photoexcitation shows migration in a protein moiety on a picosecond timescale, which is observed using time-resolved ultraviolet resonance Raman spectroscopy. The anti-Stokes ultraviolet resonance Raman intensity of a tryptophan residue is an excellent probe for the vibrational energy in proteins, allowing the mapping of energy flow with the spatial resolution of a single amino acid residue. This Perspective provides an overview of studies on vibrational energy flow in proteins, including future perspectives for both methodologies and applications.
Vibrational energy flow in the many degrees of freedom in proteins governs energy-barrier-crossing processes, such as conformational exchanges and thermal reactions. The intensity of anti-Stokes ...Raman bands arises from vibrationally excited populations and can thus function as a selective probe for the excess energy. Time-resolved observations of the anti-Stokes ultraviolet resonance Raman (UVRR) intensity of amino acid residues provide information about the flow of excess energy in proteins, with the spatial resolution of an amino acid residue. The answer to the question of whether the extent of vibrational excitation in any given vibrational modes reflects the extent of excitation in the whole molecule under nonequilibrium conditions is not straightforward. Here, we calculated the occupation probabilities of vibrational states for model compounds of amino acids under equilibrium and nonequilibrium conditions. At a given temperature, the occupation probability of the model compound of tryptophan under nonequilibrium conditions was nearly identical to that under equilibrium conditions at high temperature. Thus, the anti-Stokes band intensities of Trp residues in the nonequilibrium condition indicate the temperature of the molecules with equivalent energy in the equilibrium condition. In addition, we showed that the temperatures calculated on the basis of two UVRR bands of tryptophan in a time-resolved spectrum agreed with each other within the experimental uncertainty. The present results demonstrate that anti-Stokes UVRR bands of Trp residues serve as an excellent spectroscopic thermometer for determining the local temperature in proteins under nonequilibrium conditions.
Recent discoveries of light‐driven inward proton‐pumping rhodopsins have opened new avenues to exploring the mechanism of unidirectional transport because these proteins transport protons in the ...opposite direction to conventional proton‐pumping rhodopsins, despite their similar protein structure and membrane topology. Schizorhodopsin (SzR) is a newly discovered rhodopsin family of light‐driven inward proton pumps. Here, we report time‐resolved resonance Raman spectra showing that cis–trans thermal reisomerization precedes reprotonation at the Schiff base of the retinal chromophore in the photocycle of SzR AM_5_00977. This sequence has not been observed for the photocycles of conventional proton‐pumping rhodopsins, in which reisomerization follows reprotonation, and thus provides insights into the mechanism of proton uptake to the chromophore during inward proton pumping. The present findings are expected to contribute to controlling the direction of proton transport in engineered proteins.
A photocycle of a light‐driven inward proton‐pumping schizorhodopsin was characterized using time‐resolved resonance Raman spectroscopy, which demonstrated that cis–trans reisomerization precedes reprotonation of the retinal chromophore. This has never been observed for the conventional proton‐pumping rhodopsins. Notably, the unprecedented sequence provides insights into the mechanism of proton uptake to the chromophore during inward proton pumping.
When a chromophore embedded in a photoreceptive protein undergoes a reaction upon photoexcitation, the photoreaction triggers structural changes in the protein moiety that are necessary for the ...function of the protein. It is thus essential to elucidate the coupling between the chromophore and protein moiety to understand the functional mechanism for photoreceptive proteins, but the mechanism by which this coupling occurs remains poorly understood. Here, we show that nonbonded atomic contacts play an essential role in driving functionally important structural changes following photoisomerization of the chromophore in Gloeobacter rhodopsin (GR). Time-resolved ultraviolet resonance Raman spectroscopy revealed that the substitution of Trp222, which contacts with methyl groups of the retinal chromophore, with a Phe residue reduced the extent of structural change. The proton-pumping activity of the GR mutant was as small as 9% of that of the wild type. Time-resolved visible absorption and resonance Raman spectra showed that the photocycle of the mutant proceeded to the L intermediate following the all-trans to 13-cis photoisomerization step but did not result in the deprotonation of the chromophore. The present results demonstrate that the atomic contacts between the chromophore and the Trp222 side chain induce the structural changes necessary for proton transfer. The requirement for dense atomic packing in a protein structure for the efficient propagation of structural changes through a coupling mechanism is discussed.
We conducted a comprehensive time-resolved resonance Raman spectroscopy study of the structures of the retinal chromophore during the photocycle of the sodium-ion pump Krokinobacter rhodopsin 2 ...(KR2). We succeeded in determining the structure of the chromophore in the unphotolyzed state and in the K, L, M, and O intermediates, by overcoming the problem that only a small fraction of the M intermediate is accumulated in the KR2 photocycle. The Schiff base in the retinal chromophore forms a strong hydrogen bond in the unphotolyzed state and in the K, L, and O intermediates and is deprotonated in the M intermediate. Formation of this strong hydrogen bond facilitates deprotonation of the Schiff base, which is necessary for the sodium ion to move past the Schiff base. The polyene chain in the chromophore of KR2 is twisted in all of the states of the photocycle: the portion near the Schiff base is largely twisted in the unphotolyzed state and in the K intermediate, whereas the middle portion of the polyene chain becomes largely twisted in the L, M, and O intermediates. During the photocycle, the twisted structure of the polyene chain and strong hydrogen bond at the Schiff base are advantageous for transient relocation of the Schiff base proton. The obtained resonance Raman data clarified the unique structural features of the KR2 chromophore, which are not accessible by other methods.
Cooperativity is essential for the proper functioning of numerous proteins by allosteric interactions. Hemoglobin from Scapharca inaequivalvis (HbI) is a homodimeric protein that can serve as a ...minimal unit for studying cooperativity. We investigated the structural changes in HbI after carbon monoxide dissociation using time-resolved resonance Raman spectroscopy and observed structural rearrangements in the Fe-proximal histidine bond, the position of the heme in the pocket, and the hydrogen bonds between heme and interfacial water upon ligand dissociation. Some of the spectral changes were different from those observed for human adult hemoglobin due to differences in subunit assembly and quaternary changes. The structural rearrangements were similar for the singly and doubly dissociated species but occurred at different rates. The rates of the observed rearrangements indicated that they occurred synchronously with subunit rotation and are influenced by intersubunit coupling, which underlies the positive cooperativity of HbI.
The association and dissociation of small ligands regulate the functions of proteins through structural changes in the protein. Such structural changes propagate long distances, and this allostery ...plays a key role in molecular functions. However, the mechanism by which structural changes are transmitted is poorly understood. Here we show that nonbonded atomic contacts play an essential role in driving the displacement of a helix in picosecond time scale primary structural changes following the dissociation of carbon monoxide from the heme group in myoglobin. The present time-resolved ultraviolet resonance Raman study revealed that the amplitude of this helix displacement was reduced upon substitution of Val68, which contacts the heme in wild-type myoglobin, with a less bulky side chain (Ala). Our findings provided the first direct evidence that structural changes are transmitted not only by covalent bonds, salt bridges and hydrogen bonds but also by nonbonded atomic contacts in the primary protein response upon ligand dissociation. Furthermore, the present results indicate the importance of dense atomic packing in a protein structure for responding to the association and dissociation of small molecules. The high compactness of protein structures makes possible the propagation of structural changes, providing useful clues to the design of molecular machines.
Photoreceptor proteins play a critical role in light utilization for energy conversion and environmental sensing. Rhodopsin is a prototypical photoreceptor protein containing a retinal group that ...functions as a light-receptive site. It is essential to characterize the structure of the retinal chromophore because the chromophore structure, along with retinal–protein interactions, regulates which wavelengths of light are absorbed. Resonance Raman spectroscopy is a powerful tool to characterize chromophore structures in proteins. The resonance Raman spectra of heliorhodopsins, a recently discovered rhodopsin family, were previously reported to exhibit two intense ethylenic CC stretching bands never observed for type-1 rhodopsins. Here, we show that the double-band feature in the ethylenic CC stretching modes is not due to structural inhomogeneity but rather to the retinal polyene chain’s linear structure. It contrasts with bent all-trans chromophore in type-1 rhodopsins. The linear structure of the chromophore results from weak atomic contacts between the 13-methyl group and a nearby Trp side chain, which can slow thermal reisomerization in the photocycle. It is possible that the deceleration of reisomerization increases the lifetime of the signaling intermediate for photosensory function.
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