Elucidating interprotein energy transfer dynamics within the antenna network from purple bacteria.

In photosynthesis, absorbed light energy transfers through a network of antenna proteins with near-unity quantum efficiency to reach the reaction center, which initiates the downstream biochemical reactions. While the energy transfer dynamics within individual antenna proteins have been extensively studied over the past decades, the dynamics between the proteins are poorly understood due to the heterogeneous organization of the network. Previously reported timescales averaged over such heterogeneity, obscuring individual interprotein energy transfer steps. Here, we isolated and interrogated interprotein energy transfer by embedding two variants of the primary antenna protein from purple bacteria, light-harvesting complex 2 (LH2), together into a near-native membrane disc, known as a nanodisc. We integrated ultrafast transient absorption spectroscopy, quantum dynamics simulations, and cryogenic electron microscopy to determine interprotein energy transfer timescales. By varying the diameter of the nanodiscs, we replicated a range of distances between the proteins. The closest distance possible between neighboring LH2, which is the most common in native membranes, is 25 Šand resulted in a timescale of 5.7 ps. Larger distances of 28 to 31 Šresulted in timescales of 10 to 14 ps. Corresponding simulations showed that the fast energy transfer steps between closely spaced LH2 increase transport distances by ∼15%. Overall, our results introduce a framework for well-controlled studies of interprotein energy transfer dynamics and suggest that protein pairs serve as the primary pathway for the efficient transport of solar energy.

Proton transfers in a channelrhodopsin-1 studied by Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis.

Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs.

Comparison of the structural changes occurring during the primary phototransition of two different channelrhodopsins from Chlamydomonas algae.

Channelrhodopsins (ChRs) from green flagellate algae function as light-gated ion channels when expressed heterologously in mammalian cells. Considerable interest has focused on understanding the molecular mechanisms of ChRs to bioengineer their properties for specific optogenetic applications such as elucidating the function of specific neurons in brain circuits. While most studies have used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), in this work low-temperature Fourier transform infrared-difference spectroscopy is applied to study the conformational changes occurring during the primary phototransition of the red-shifted ChR1 from Chlamydomonas augustae (CaChR1). Substitution with isotope-labeled retinals or the retinal analogue A2, site-directed mutagenesis, hydrogen-deuterium exchange, and H2(18)O exchange were used to assign bands to the retinal chromophore, protein, and internal water molecules. The primary phototransition of CaChR1 at 80 K involves, in contrast to that of CrChR2, almost exclusively an all-trans to 13-cis isomerization of the retinal chromophore, as in the primary phototransition of bacteriorhodopsin (BR). In addition, significant differences are found for structural changes of the protein and internal water(s) compared to those of CrChR2, including the response of several Asp/Glu residues to retinal isomerization. A negative amide II band is identified in the retinal ethylenic stretch region of CaChR1, which reflects along with amide I bands alterations in protein backbone structure early in the photocycle. A decrease in the hydrogen bond strength of a weakly hydrogen bonded internal water is detected in both CaChR1 and CrChR2, but the bands are much broader in CrChR2, indicating a more heterogeneous environment. Mutations involving residues Glu169 and Asp299 (homologues of the Asp85 and Asp212 Schiff base counterions, respectively, in BR) lead to the conclusion that Asp299 is protonated during P1 formation and suggest that these residues interact through a strong hydrogen bond that facilitates the transfer of a proton from Glu169.

Retinal chromophore structure and Schiff base interactions in red-shifted channelrhodopsin-1 from Chlamydomonas augustae.

Channelrhodopsins (ChRs), which form a distinct branch of the microbial rhodopsin family, control phototaxis in green algae. Because ChRs can be expressed and function in neuronal membranes as light-gated cation channels, they have rapidly become an important optogenetic tool in neurobiology. While channelrhodopsin-2 from the unicellular alga Chlamydomonas reinhardtii (CrChR2) is the most commonly used and extensively studied optogenetic ChR, little is known about the properties of the diverse group of other ChRs. In this study, near-infrared confocal resonance Raman spectroscopy along with hydrogen-deuterium exchange and site-directed mutagenesis were used to study the structure of red-shifted ChR1 from Chlamydomonas augustae (CaChR1). These measurements reveal that (i) CaChR1 has an all-trans-retinal structure similar to those of the light-driven proton pump bacteriorhodopsin (BR) and sensory rhodopsin II but different from that of the mixed retinal composition of CrChR2, (ii) lowering the pH from 7 to 2 or substituting neutral residues for Glu169 or Asp299 does not significantly shift the ethylenic stretch frequency more than 1-2 cm(-1) in contrast to BR in which a downshift of 7-9 cm(-1) occurs reflecting neutralization of the Asp85 counterion, and (iii) the CaChR1 protonated Schiff base (SB) has stronger hydrogen bonding than BR. A model is proposed to explain these results whereby at pH 7 the predominant counterion to the SB is Asp299 (the homologue to Asp212 in BR) while Glu169 (the homologue to Asp85 in BR) exists in a neutral state. We observe an unusual constancy of the resonance Raman spectra over the broad range from pH 9 to 2 and discuss its implications. These results are in accord with recent visible absorption and current measurements of CaChR1 [Sineshchekov, O. A., et al. (2013) Intramolecular proton transfer in channelrhodopsins. Biophys. J. 104, 807-817; Li, H., et al. (2014) Role of a helix B lysine residue in the photoactive site in channelrhodopsins. Biophys. J. 106, 1607-1617].

High-Density, Actively Multiplexed µECoG Array on Reinforced Silicone Substrate.

Simultaneous interrogation of electrical signals from wide areas of the brain is vital for neuroscience research and can aid in understanding the mechanisms of brain function and treatments for neurological disorders. There emerges a demand for development of devices with highly conformal interfaces that can span large cortical regions, have sufficient spatial resolution, and chronic recording capability while keeping a small implantation footprint. In this work, we have designed 61 channel and 48 channel high-density, cortical, micro-electrocorticographic electrode arrays with 400 µm pitch on an ultra-soft but durable substrate. We have also developed a custom multiplexing integrated circuit (IC), methods for packaging the IC in a water-tight liquid crystal polymer casing, and a micro-bonding method for attaching the electronics package to the electrode array. With the integrated multiplexer, the number of external wire connections can be reduced to 16 wires, thereby diminishing the invasive footprint of the device. Both the electrode array and IC were tested in vivo in a rat model to demonstrate the ability to sense finely-localized electrophysiological signals.

High-density spinal cord stimulation selectively activates lower urinary tract nerves.

Objective.Epidural spinal cord stimulation (SCS) is a potential intervention to improve limb and autonomic functions, with lumbar stimulation improving locomotion and thoracic stimulation regulating blood pressure. Here, we asked whether sacral SCS could be used to target the lower urinary tract (LUT) and used a high-density epidural electrode array to test whether individual electrodes could selectively recruit LUT nerves.Approach. We placed a high-density epidural SCS array on the dorsal surface of the sacral spinal cord and cauda equina of anesthetized cats and recorded the stimulation-evoked activity from nerve cuffs on the pelvic, pudendal and sciatic nerves.Main results. Here we show that sacral SCS evokes responses in nerves innervating the bladder and urethra and that these nerves can be activated selectively. Sacral SCS always recruited the pelvic and pudendal nerves and selectively recruited both of these nerves in all but one animal. Individual branches of the pudendal nerve were always recruited as well. Electrodes that selectively recruited specific peripheral nerves were spatially clustered on the arrays, suggesting anatomically organized sensory pathways.Significance.This selective recruitment demonstrates a mechanism to directly modulate bladder and urethral function through known reflex pathways, which could be used to restore bladder and urethral function after injury or disease.

Impact of the lipid bilayer on energy transfer kinetics in the photosynthetic protein LH2.

Photosynthetic purple bacteria convert solar energy to chemical energy with near unity quantum efficiency. The light-harvesting process begins with absorption of solar energy by an antenna protein called Light-Harvesting Complex 2 (LH2). Energy is subsequently transferred within LH2 and then through a network of additional light-harvesting proteins to a central location, termed the reaction center, where charge separation occurs. The energy transfer dynamics of LH2 are highly sensitive to intermolecular distances and relative organizations. As a result, minor structural perturbations can cause significant changes in these dynamics. Previous experiments have primarily been performed in two ways. One uses non-native samples where LH2 is solubilized in detergent, which can alter protein structure. The other uses complex membranes that contain multiple proteins within a large lipid area, which make it difficult to identify and distinguish perturbations caused by protein-protein interactions and lipid-protein interactions. Here, we introduce the use of the biochemical platform of model membrane discs to study the energy transfer dynamics of photosynthetic light-harvesting complexes in a near-native environment. We incorporate a single LH2 from Rhodobacter sphaeroides into membrane discs that provide a spectroscopically amenable sample in an environment more physiological than detergent but less complex than traditional membranes. This provides a simplified system to understand an individual protein and how the lipid-protein interaction affects energy transfer dynamics. We compare the energy transfer rates of detergent-solubilized LH2 with those of LH2 in membrane discs using transient absorption spectroscopy and transient absorption anisotropy. For one key energy transfer step in LH2, we observe a 30% enhancement of the rate for LH2 in membrane discs compared to that in detergent. Based on experimental results and theoretical modeling, we attribute this difference to tilting of the peripheral bacteriochlorophyll in the B800 band. These results highlight the importance of well-defined systems with near-native membrane conditions for physiologically-relevant measurements.

Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals.

Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump-probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals.

7 Å resolution in protein two-dimensional-crystal X-ray diffraction at Linac Coherent Light Source.

Membrane proteins arranged as two-dimensional crystals in the lipid environment provide close-to-physiological structural information, which is essential for understanding the molecular mechanisms of protein function. Previously, X-ray diffraction from individual two-dimensional crystals did not represent a suitable investigational tool because of radiation damage. The recent availability of ultrashort pulses from X-ray free-electron lasers (XFELs) has now provided a means to outrun the damage. Here, we report on measurements performed at the Linac Coherent Light Source XFEL on bacteriorhodopsin two-dimensional crystals mounted on a solid support and kept at room temperature. By merging data from about a dozen single crystal diffraction images, we unambiguously identified the diffraction peaks to a resolution of 7 Å, thus improving the observable resolution with respect to that achievable from a single pattern alone. This indicates that a larger dataset will allow for reliable quantification of peak intensities, and in turn a corresponding increase in the resolution. The presented results pave the way for further XFEL studies on two-dimensional crystals, which may include pump-probe experiments at subpicosecond time resolution.

Conformational changes in the archaerhodopsin-3 proton pump: detection of conserved strongly hydrogen bonded water networks.

Archaerhodopsin-3 (AR3) is a light-driven proton pump from Halorubrum sodomense, but little is known about its photocycle. Recent interest has focused on AR3 because of its ability to serve both as a high-performance, genetically-targetable optical silencer of neuronal activity and as a membrane voltage sensor. We examined light-activated structural changes of the protein, retinal chromophore, and internal water molecules during the photocycle of AR3. Low-temperature and rapid-scan time-resolved FTIR-difference spectroscopy revealed that conformational changes during formation of the K, M, and N photocycle intermediates are similar, although not identical, to bacteriorhodopsin (BR). Positive/negative bands in the region above 3,600 cm( - 1), which have previously been assigned to structural changes of weakly hydrogen bonded internal water molecules, were substantially different between AR3 and BR. This included the absence of positive bands recently associated with a chain of proton transporting water molecules in the cytoplasmic channel and a weakly hydrogen bonded water (W401), which is part of a hydrogen-bonded pentagonal cluster located near the retinal Schiff base. However, many of the broad IR continuum absorption changes below 3,000 cm( - 1) assigned to networks of water molecules involved in proton transport through cytoplasmic and extracellular portions in BR were very similar in AR3. This work and subsequent studies comparing BR and AR3 structural changes will help identify conserved elements in BR-like proton pumps as well as bioengineer AR3 to optimize neural silencing and voltage sensing.

Near-IR resonance Raman spectroscopy of archaerhodopsin 3: effects of transmembrane potential.

Archaerhodopsin 3 (AR3) is a light driven proton pump from Halorubrum sodomense that has been used as a genetically targetable neuronal silencer and an effective fluorescent sensor of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 readily incorporates into the plasma membrane of both E. coli and mammalian cells. Here, we used near-IR resonance Raman confocal microscopy to study the effects of pH and membrane potential on the AR3 retinal chromophore structure. Measurements were performed both on AR3 reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of a negative transmembrane potential. The retinal chromophore structure of AR3 is in an all-trans configuration almost identical to BR over the entire pH range from 3 to 11. Small changes are detected in the retinal ethylenic stretching frequency and Schiff Base (SB) hydrogen bonding strength relative to BR which may be related to a different water structure near the SB. In the case of the AR3 mutant D95N, at neutral pH an all-trans retinal O-like species (O(all-trans)) is found. At higher pH a second 13-cis retinal N-like species (N(13-cis)) is detected which is attributed to a slowly decaying intermediate in the red-light photocycle of D95N. However, the amount of N(13-cis) detected is less in E. coli cells but is restored upon addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or sonication, both of which dissipate the normal negative membrane potential. We postulate that these changes are due to the effect of membrane potential on the N(13-cis) to M(13-cis) levels accumulated in the D95N red-light photocycle and on a molecular level by the effects of the electric field on the protonation/deprotonation of the cytoplasmic accessible SB. This mechanism also provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential.