Sub-Tip-Radius Near-Field Interactions in Nano-FTIR Vibrational Spectroscopy on Single Proteins.

Tip-enhanced vibrational spectroscopy has advanced to routinely attain nanoscale spatial resolution, with tip-enhanced Raman spectroscopy even achieving atomic-scale and submolecular sensitivity. Tip-enhanced infrared spectroscopy techniques, such as nano-FTIR and AFM-IR spectroscopy, have also enabled the nanoscale chemical analysis of molecular monolayers, inorganic nanoparticles, and protein complexes. However, fundamental limits of infrared nanospectroscopy in terms of spatial resolution and sensitivity have remained elusive, calling for a quantitative understanding of the near-field interactions in infrared nanocavities. Here, we demonstrate the application of nano-FTIR spectroscopy to probe the amide-I vibration of a single protein consisting of ∼500 amino acid residues. Detection with higher tip tapping demodulation harmonics up to the seventh order leads to pronounced enhancement in the peak amplitude of the vibrational resonance, originating from sub-tip-radius geometrical effects beyond dipole approximations. This quantitative characterization of single-nanometer near-field interactions opens the path toward employing infrared vibrational spectroscopy at the subnanoscale and single-molecule levels.

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

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.

Direct observation of stepping rotation of V-ATPase reveals rigid component in coupling between Vo and V1 motors.

V-ATPases are rotary motor proteins that convert the chemical energy of ATP into the electrochemical potential of ions across cell membranes. V-ATPases consist of two rotary motors, Vo and V1, and Enterococcus hirae V-ATPase (EhVoV1) actively transports Na+ in Vo (EhVo) by using torque generated by ATP hydrolysis in V1 (EhV1). Here, we observed ATP-driven stepping rotation of detergent-solubilized EhVoV1 wild-type, aE634A, and BR350K mutants under various Na+ and ATP concentrations ([Na+] and [ATP], respectively) by using a 40-nm gold nanoparticle as a low-load probe. When [Na+] was low and [ATP] was high, under the condition that only Na+ binding to EhVo is rate limiting, wild-type and aE634A exhibited 10 pausing positions reflecting 10-fold symmetry of the EhVo rotor and almost no backward steps. Duration time before the forward steps was inversely proportional to [Na+], confirming that Na+ binding triggers the steps. When both [ATP] and [Na+] were low, under the condition that both Na+ and ATP bindings are rate limiting, aE634A exhibited 13 pausing positions reflecting 10- and 3-fold symmetries of EhVo and EhV1, respectively. The distribution of duration time before the forward step was fitted well by the sum of two exponential decay functions with distinct time constants. Furthermore, occasional backward steps smaller than 36° were observed. Small backward steps were also observed during three long ATP cleavage pauses of BR350K. These results indicate that EhVo and EhV1 do not share pausing positions, Na+ and ATP bindings occur at different angles, and the coupling between EhVo and EhV1 has a rigid component.

Resonance Raman Determination of Chromophore Structures of Heliorhodopsin Photointermediates.

Light is utilized as energy or information by rhodopsins (membrane proteins that contain a retinal chromophore). Heliorhodopsins (HeRs) are a new class of rhodopsins with low sequence identity (<15%) to microbial and animal rhodopsins. Their physiological roles remain unknown, although the involvement of a long-lived intermediate in the photocycle suggests a light-sensor function. Characterization of the molecular structures of the intermediates is essential to an understanding of the roles and mechanisms of HeRs. We determined the chromophore structures of the intermediates in HeR 48C12 by time-resolved resonance Raman spectroscopy and observed that the hydrogen bond of the protonated Schiff base strengthened prior to deprotonation. The chromophore is photoisomerized from the all-trans to the 13-cis form and is reisomerized in the transition from the O intermediate to the unphotolyzed state. Our results demonstrate that the chromophore structure evolves similarly to microbial rhodopsins, despite the dissimilarity in amino acid residues surrounding the chromophore.

Allosteric Communication with the Retinal Chromophore upon Ion Binding in a Light-Driven Sodium Ion-Pumping Rhodopsin.

Krokinobacter rhodopsin 2 (KR2) serves as a light-driven sodium ion pump in the presence of sodium ion and works as a proton pump in the presence of larger monovalent cations such as potassium ion, rubidium ion, and cesium ion. Recent crystallographic studies revealed that KR2 forms a pentamer and possesses an ion binding site at the subunit interface. It is assumed that sodium ion bound at this binding site is not transported but contributes to the thermal stability. Because KR2 can convert its function in response to coexisting cation species, this ion binding site is likely to be involved in ion transport selectively. However, how sodium ion binding affects the structure of the retinal chromophore, which plays a crucial role in ion transport, remains poorly understood. Here, we observed the structure of the retinal chromophore under a wide range of cation concentrations using visible absorption and resonance Raman spectroscopy. We discovered that the hydrogen bond formed between the Schiff base of the retinal chromophore and its counterion, Asp116, is weakened upon binding of sodium ion. This allosteric communication between the Schiff base and the ion binding site at the subunit interface likely increases the apparent efficiency of sodium ion transport. In addition, this study demonstrates the significance of sodium ion binding: even though sodium ion is not transported, binding regulates the structure around the Schiff base and stabilizes the oligomeric structure.

Resonance Raman Investigation of the Chromophore Structure of Heliorhodopsins.

Heliorhodopsins (HeRs) are a new category of retinal-bound proteins recently discovered through functional metagenomics analysis that exhibit obvious differences from type-1 microbial rhodopsins. We conducted the first detailed structural characterization of the retinal chromophore in HeRs using resonance Raman spectroscopy. The observed spectra clearly show that the Schiff base of the chromophore is protonated and forms a strong hydrogen bond to a species other than a water molecule, highly likely a counterion residue. The vibrational mode of the Schiff base of HeRs exhibits similarities with that of photosensory microbial rhodopsins, that is consistent with the previous proposal that HeRs function as photosensors. We also revealed unusual spectral features of the in-plane chain vibrations of the chromophore, suggesting an unprecedented geometry of the Schiff base caused by a difference in the retinal pocket structure of HeRs. These data demonstrate structural characteristics of the photoreceptive site in this novel type of rhodopsin family.

A Study of the Dynamics of the Heme Pocket and C-helix in CooA upon CO Dissociation Using Time-Resolved Visible and UV Resonance Raman Spectroscopy.

CooA is a CO-sensing transcriptional activator from the photosynthetic bacterium Rhodospirillum rubrum that binds CO at the heme iron. The heme iron in ferrous CooA has two axial ligands: His77 and Pro2. CO displaces Pro2 and induces a conformational change in CooA. The dissociation of CO and/or ligation of the Pro2 residue are believed to trigger structural changes in the protein. Visible time-resolved resonance Raman spectra obtained in this study indicated that the ν(Fe-His) mode, arising from the proximal His77-iron stretch, does not shift until 50 µs after the photodissociation of CO. Ligation of the Pro2 residue to the heme iron was observed around 50 µs after the photodissociation of CO, suggesting that the ν(Fe-His) band exhibits no shift until the ligation of Pro2. UV resonance Raman spectra suggested structural changes in the vicinity of Trp110 in the C-helix upon CO binding, but no or very small spectral changes in the time-resolved UV resonance Raman spectra were observed from 100 ns to 100 µs after the photodissociation of CO. These results strongly suggest that the conformational change of CooA is induced by the ligation of Pro2 to the heme iron.