Parallel photocycle kinetic model of anion channelrhodopsin GtACR1 function.

The light-gated anion channelrhodopsin GtACR1 is an important optogenetic tool for neuronal silencing. Its photochemistry, including its photointermediates, is poorly understood. The current mechanistic view presumes BR-like kinetics and assigns the open channel to a blue-absorbing L intermediate. Based on time-resolved absorption and electrophysiological data, we recently proposed a red-absorbing spectral form for the open channel state. Here, we report the results of a comprehensive kinetic analysis of the spectroscopic data combined with channel current information. The time evolutions of the spectral forms derived from the spectroscopic data are inconsistent with the single chain mechanism and are analyzed within the concept of parallel photocycles. The spectral forms partitioned into conductive and nonconductive parallel cycles are assigned to intermediate states. Rejecting reversible connections between conductive and nonconductive channel states leads to kinetic schemes with two independent conductive states corresponding to the fast- and slow-decaying current components. The conductive cycle is discussed in terms of a single cycle and two parallel cycles. The reaction mechanisms and reaction rates for the wild-type protein, the A75E, and the low-conductance D234N and S97E protein variants are derived. The parallel cycles of channelrhodopsin kinetics, its relation to BR photocycle, and the role of the M intermediate in channel closure are discussed.

The open channel state in anion channelrhodopsin GtACR1 is a red-absorbing intermediate.

Anion channelrhodopsin GtACR1 is a powerful optogenetic tool to inhibit nerve activity. Its kinetic mechanism was interpreted in terms of the bacteriorhodopsin photocycle, and the L intermediate was assigned to the open channel state. Here, we report the results of the comparison between the time dependence of the channel currents and the time evolutions of the K-like and L-like spectral forms. Based on the results, we question the current view on GtACR1 kinetics and the assignment of the L intermediate to the open channel state. We report evidence for a red-absorbing intermediate being responsible for channel opening.

Isospectral intermediates in the photochemical reaction cycle of anion channelrhodopsin GtACR1.

The most effective tested optogenetic tools available for neuronal silencing are the light-gated anion channel proteins found in the cryptophyte alga Guillardia theta (GtACRs). Molecular mechanisms of GtACRs, including the photointermediates responsible for the open channel state, are of great interest for understanding their exceptional conductance. In this study, the photoreactions of GtACR1 and its D234N, A75E, and S97E mutants were investigated using multichannel time-resolved absorption spectroscopy. For each of the proteins, the analysis showed two early microsecond transitions between K-like and L-like forms and two late millisecond recovery steps. Spectral forms associated with potential molecular intermediates of the proteins were derived and their evolutions in time were analyzed. The results indicate the presence of isospectral intermediates in the photocycles and expand the range of potential intermediates responsible for the open channel state.

Styrene-maleic acid copolymer effects on the function of the GPCR rhodopsin in lipid nanoparticles.

Styrene-maleic acid (SMA) copolymers solubilize biological membranes to form lipid nanoparticles (SMALPs) that contain membrane proteins surrounded by native lipids, thus enabling the use of a variety of biophysical techniques for structural and functional studies. The question of whether SMALPs provide a truly natural environment or SMA solubilization affects the functional properties of membrane proteins, however, remains open. We address this question by comparing the photoactivation kinetics of rhodopsin, a G-protein-coupled receptor in the disk membranes of rod cells, in native membrane and SMALPs prepared at different molar ratios between SMA(3:1) and rhodopsin. Time-resolved absorption spectroscopy combined with complex kinetic analysis reveals kinetic and mechanistic differences between the native membrane and SMA-stabilized environment. The results suggest a range of molar ratios for nanoparticles suitable for kinetic studies.

Functional integrity of membrane protein rhodopsin solubilized by styrene-maleic acid copolymer.

Membrane proteins often require solubilization to study their structure or define the mechanisms underlying their function. In this study, the functional properties of the membrane protein rhodopsin in its native lipid environment were investigated after being solubilized with styrene-maleic acid (SMA) copolymer. The static absorption spectra of rhodopsin before and after the addition of SMA were recorded at room temperature to quantify the amount of membrane protein solubilized. The samples were then photobleached to analyze the functionality of rhodopsin upon solubilization. Samples with low or high SMA/rhodopsin ratios were compared to find a threshold in which the maximal amount of active rhodopsin was solubilized from membrane suspensions. Interestingly, whereas the highest SMA/rhodopsin ratios yielded the most solubilized rhodopsin, the rhodopsin produced under these conditions could not reach the active (Meta II) state upon photoactivation. The results confirm that SMA is a useful tool for membrane protein research, but SMA added in excess can interfere with the dynamics of protein activation.

Pump-Probe Circular Dichroism Spectroscopy of Cyanobacteriochrome TePixJ Yields: Insights into Its Photoconversion.

The bilin-containing photoreceptor TePixJ, a member of the cyanobacteriochrome (CBCR) family of phytochromes, switches between blue-light-absorbing and green-light-absorbing states in order to drive phototaxis in Thermosynechococcus elongatus. Its photoswitching process involves the formation of a thioether linkage between the C10 carbon of phycoviolobilin and the sidechain of Cys494 during the change in state from green-absorbing to blue-absorbing forms. Complex changes in the binding pocket propagate the signal to other domains for downstream signaling. Here, we report time-resolved circular dichroism experiments in addition to pump-probe absorption measurements for interpretation of the biophysical mechanism of the green-to-blue photoconversion process of this receptor.

Membrane Curvature Revisited-the Archetype of Rhodopsin Studied by Time-Resolved Electronic Spectroscopy.

G-protein-coupled receptors (GPCRs) comprise the largest and most pharmacologically targeted membrane protein family. Here, we used the visual receptor rhodopsin as an archetype for understanding membrane lipid influences on conformational changes involved in GPCR activation. Visual rhodopsin was recombined with lipids varying in their degree of acyl chain unsaturation and polar headgroup size using 1-palmitoyl-2-oleoyl-sn-glycero- and 1,2-dioleoyl-sn-glycerophospholipids with phosphocholine (PC) or phosphoethanolamine (PE) substituents. The receptor activation profile after light excitation was measured using time-resolved ultraviolet-visible spectroscopy. We discovered that more saturated POPC lipids back shifted the equilibrium to the inactive state, whereas the small-headgroup, highly unsaturated DOPE lipids favored the active state. Increasing unsaturation and decreasing headgroup size have similar effects that combine to yield control of rhodopsin activation, and necessitate factors beyond proteolipid solvation energy and bilayer surface electrostatics. Hence, we consider a balance of curvature free energy with hydrophobic matching and demonstrate how our data support a flexible surface model (FSM) for the coupling between proteins and lipids. The FSM is based on the Helfrich formulation of membrane bending energy as we previously first applied to lipid-protein interactions. Membrane elasticity and curvature strain are induced by lateral pressure imbalances between the constituent lipids and drive key physiological processes at the membrane level. Spontaneous negative monolayer curvature toward water is mediated by unsaturated, small-headgroup lipids and couples directly to GPCR activation upon light absorption by rhodopsin. For the first time to our knowledge, we demonstrate this modulation in both the equilibrium and pre-equilibrium evolving states using a time-resolved approach.

First Synthesis of Mn-Doped Cesium Lead Bromide Perovskite Magic Sized Clusters at Room Temperature.

Mn-doped CsPbBr3 perovskite magic sized clusters (PMSCs) are synthesized for the first time using benzoic acid and benzylamine as passivating ligands and MnCl2·4H2O and MnBr2 as the Mn2+ dopant sources at room temperature. The same approach is used to prepare Mn-doped CsPbBr3 perovskite quantum dots (PQDs). The concentration of MnX2 (X = Cl or Br) affects the excitonic absorption of the PMSCs and PQDs. A higher concentration of MnX2 favors PMSCs over PQDs as well as higher photoluminescence (PL) quantum yields (QYs) and PL stability. The large ratio between the characteristic Mn emission (∼590 nm) and the host band-edge emission shows efficient energy transfer from the host exciton to the Mn2+ dopant. PL excitation, electron paramagnetic resonance, and time-resolved PL results all support Mn2+ doping in CsPbBr3, which likely replaces Pb2+ ions. This study establishes a new method for synthesizing Mn-doped PMSCs with good PL stability, high PLQY and highly effective passivation.

VenusA206 Dimers Behave Coherently at Room Temperature.

Fluorescent proteins (FPs) have revolutionized cell biology by allowing genetic tagging of specific proteins inside living cells. In conjunction with Förster's resonance energy transfer (FRET) measurements, FP-tagged proteins can be used to study protein-protein interactions and estimate distances between tagged proteins. FRET is mediated by weak Coulombic dipole-dipole coupling of donor and acceptor fluorophores that behave independently, with energy hopping discretely and incoherently between fluorophores. Stronger dipole-dipole coupling can mediate excitonic coupling in which excitation energy is distributed near instantaneously between coherently interacting excited states that behave as a single quantum entity. The interpretation of FP energy transfer measurements to estimate separation often assumes that donors and acceptors are very weakly coupled and therefore use a FRET mechanism. This assumption is considered reasonable as close fluorophore proximity, typically associated with strong excitonic coupling, is limited by the FP ß-barrel structure. Furthermore, physiological temperatures promote rapid vibrational dephasing associated with a rapid decoherence of fluorophore-excited states. Recently, FP dephasing times that are 50 times slower than traditional organic fluorophores have been measured, raising the possibility that evolution has shaped FPs to allow stronger than expected coupling under physiological conditions. In this study, we test if excitonic coupling between FPs is possible at physiological temperatures. FRET and excitonic coupling can be distinguished by monitoring spectral changes associated with fluorophore dimerization. The weak coupling mediating FRET should not cause a change in fluorophore absorption, whereas strong excitonic coupling causes Davydov splitting. Circular dichroism spectroscopy revealed Davydov splitting when the yellow FP VenusA206 dimerizes, and a novel approach combining photon antibunching and fluorescence correlation spectroscopy was used to confirm that the two fluorophores in a VenusA206 homodimer behave as a single-photon emitter. We conclude that excitonic coupling between VenusA206 fluorophores is possible at physiological temperatures.

Microviscosity in E. coli Cells from Time-Resolved Linear Dichroism Measurements.

A protein's folding or function depends on its mobility through the viscous environment that is defined by the presence of macromolecules throughout the cell. The relevant parameter for this mobility is microviscosity-the viscosity on a time and distance scale that is important for protein folding/function movements. A quasi-null, ultrasensitive time-resolved linear dichroism (TRLD) spectroscopy is proving to be a useful tool for measurements of viscosity on this scale, with previous in vitro studies reporting on the microviscosities of crowded environments mimicked by high concentrations of different macromolecules. This study reports the microviscosity experienced by myoglobin in the E. coli cell's heterogeneous cytoplasm by using TRLD to measure rotational diffusion times. The results show that photolyzed deoxyMb ensembles randomize through environment-dependent rotational diffusion with a lifetime of 34 ± 6 ns. This value corresponds to a microviscosity of 2.82 ± 0.42 cP, which is consistent with previous reports of cytoplasmic viscosity in E. coli. The results of these TRLD studies in E. coli (1) provide a measurement of myoglobin mobility in the cytoplasm, (2) taken together with in vitro TRLD studies yield new insights into the nature of the cytoplasmic environment in cells, and (3) demonstrate the feasibility of TRLD as a probe of intracellular viscosity.

Protein Sequence and Membrane Lipid Roles in the Activation Kinetics of Bovine and Human Rhodopsins.

Rhodopsin is a G protein-coupled receptor found in the rod outer segments in the retina, which triggers a visual response under dim light conditions. Recently, a study of the late, microsecond-to-millisecond kinetics of photointermediates of the human and bovine rhodopsins in their native membranes revealed a complex, double-square mechanism of rhodopsin activation. In this kinetic scheme, the human rhodopsin exhibited more Schiff base deprotonation than bovine rhodopsin, which could arise from the ∼7% sequence difference between the two proteins, or from the difference between their membrane lipid environments. To differentiate between the effects of membrane and protein structure on the kinetics, the human and bovine rhodopsins were inserted into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid nanodiscs and the kinetics of activation at 15°C and pH 8.7 was investigated by time-resolved absorption spectroscopy and global kinetic analysis. For both proteins, the kinetics in nanodiscs shows the characteristics observed in the native membranes, and is described by a multisquare model with Schiff base deprotonation at the lumirhodopsin I intermediate stage. The results indicate that the protein sequence controls the extent of Schiff base deprotonation and accumulation of intermediates, and thus plays the main role in the different activation kinetics observed between human and bovine rhodopsins. The membrane lipid does have a minor role by modulating the timing of the kinetics, with the nanodisc environment leading to an earlier Schiff base deprotonation.

Time-Resolved Linear Dichroism Measurements of Carbonmonoxy Myoglobin as a Probe of the Microviscosity in Crowded Environments.

The distribution of viscosities in living cells is heterogeneous because of the different sizes and natures of macromolecular components. When thinking about protein folding/function processes in such an environment, the relevant (micro)viscosity at the micrometer length scale is necessarily distinguished from the bulk (macro)viscosity. The concentration dependencies of microviscosities are determined by a number of factors, such as electrostatic interactions, van der Waals forces, and excluded volume effects. To explore such factors, the rotational diffusion time of myoglobin in the presence of varying concentrations of macromolecules that differ in molecular weight (dextran 6000, 10 000, and 70 000), shape (dextran versus Ficoll), size, and surface charge is measured with time-resolved linear dichroism spectroscopy. The results of these studies offer simple empirically determined linear and exponential functions useful for predicting microviscosities as a function of concentration for these macromolecular crowders that are typically used to study crowding effects on protein folding. To understand how relevant these microviscosity measurements are to intracellular environments, the TRLD results are discussed in the context of studies that measure viscosity in cells.

Using chiral peptide substitutions to probe the structure function relationship of a key residue of Aß42.

Amyloid beta-protein 42 plays an important role in the onset and progression of Alzheimer's disease. Familial mutations have identified the glutamate residue 22 as a hotspot with regard to peptide neurotoxicity. We introduce an approach to study the influence of systematic sidechain modification at this residue, employing chirality as a structural probe. Circular dichroism experiments reveal that charge-preserving alterations of the amino acid sidechain attenuate the characteristic random coil to ß-sheet transition associated with the wildtype peptide. Removal of the negative charge from residue 22, a trait observed with all known familial mutations at this residue, gives rise to a peptide with limited random coil propensity and high ß-sheet characteristics. Our approach can be extended to other residues of Aß, as well as further amyloidogenic peptides.

A Comparison between the Photoactivation Kinetics of Human and Bovine Rhodopsins.

Rhodopsin is a G-protein-coupled receptor important for vertebrate vision under dim light conditions. Many studies of the activation mechanism of bovine rhodopsin have been conducted, but there have been relatively few investigations of the human protein. A recent study of the late photointermediates of bovine rhodopsin studies at 15 °C and pH 7.3, 8.0, and 8.7 revealed a rather complex activation mechanism involving two metarhodopsin I480 and metarhodopsin II intermediates. Human rhodopsin was studied under these same conditions using time-resolved optical absorption spectroscopy with measurements from 10 µs to 200 ms after photolysis. The results show that the two proteins follow the same photoactivation mechanism, although their kinetics differ significantly. The comparison of bovine and human rhodopsins shows that the initial Schiff base deprotonation equilibrium is more forward shifted in human rhodopsin, and more of the reaction flows through the metarhodopsin I380 intermediate in human rhodopsin than in the bovine protein.

Complexity of Bovine Rhodopsin Activation Revealed at Low Temperature and Alkaline pH.

The late intermediates involved in the activation mechanism of bovine rhodopsin are investigated by time-resolved optical absorption spectroscopy. Measurements from 10 µs to 200 ms after photolysis were carried out on membrane suspensions of bovine rhodopsin at a temperature of 15 °C and at pH of 7.3, 8.0, and 8.7. The time-resolved absorption spectra in the 330-650 nm range were analyzed by global exponential and kinetic scheme fitting methods. The results indicate an activation mechanism that is more complex than suggested previously. It involves interconnected branched pathways with two metarhodopsin I480 and two metarhodopsin II intermediates. The intermediates involved in this more complex mechanism need to be considered in spectroscopic studies that vary sample temperature and pH in order to enhance the presence of specific rhodopsin intermediates.

Role of Heme Pocket Water in Allosteric Regulation of Ligand Reactivity in Human Hemoglobin.

Water molecules can enter the heme pockets of unliganded myoglobins and hemoglobins, hydrogen bond with the distal histidine, and introduce steric barriers to ligand binding. The spectrokinetics of photodissociated CO complexes of human hemoglobin and its isolated α and ß chains were analyzed for the effect of heme hydration on ligand rebinding. A strong coupling was observed between heme hydration and quaternary state. This coupling may contribute significantly to the 20-60-fold difference between the R- and T-state bimolecular CO binding rate constants and thus to the modulation of ligand reactivity that is the hallmark of hemoglobin allostery. Heme hydration proceeded over the course of several kinetic phases in the tetramer, including the R to T quaternary transition. An initial 150 ns hydration phase increased the R-state distal pocket water occupancy, nw(R), to a level similar to that of the isolated α (∼60%) and ß (∼10%) chains, resulting in a modest barrier to ligand binding. A subsequent phase, concurrent with the first step of the R → T transition, further increased the level of heme hydration, increasing the barrier. The final phase, concurrent with the final step of the allosteric transition, brought the water occupancy of the T-state tetramer, nw(T), even higher and close to full occupancy in both the α and ß subunits (∼90%). This hydration level could present an even larger barrier to ligand binding and contribute significantly to the lower iron reactivity of the T state toward CO.

Platymonas subcordiformis Channelrhodopsin-2 Function: I. THE PHOTOCHEMICAL REACTION CYCLE.

The photocycle kinetics of Platymonas subcordiformis channelrhodopsin-2 (PsChR2), among the most highly efficient light-gated cation channels and the most blue-shifted channelrhodopsin, was studied by time-resolved absorption spectroscopy in the 340-650-nm range and in the 100-ns to 3-s time window. Global exponential fitting of the time dependence of spectral changes revealed six lifetimes: 0.60 µs, 5.3 µs, 170 µs, 1.4 ms, 6.7 ms, and 1.4 s. The sequential intermediates derived for a single unidirectional cycle scheme based on these lifetimes were found to contain mixtures of K, L, M, O, and P molecular states, named in analogy to photointermediates in the bacteriorhodopsin photocycle. The photochemistry is described by the superposition of two independent parallel photocycles. The analysis revealed that 30% of the photoexcited receptor molecules followed Cycle 1 through the K, M, O, and P states, whereas 70% followed Cycle 2 through the K, L, M, and O states. The recovered state, R, is spectrally close, but not identical, to the dark state on the seconds time scale. The two-cycle model of this high efficiency channelrhodopsin-2 (ChR) opens new perspectives in understanding the mechanism of channelrhodopsin function.

Platymonas subcordiformis Channelrhodopsin-2 (PsChR2) Function: II. RELATIONSHIP OF THE PHOTOCHEMICAL REACTION CYCLE TO CHANNEL CURRENTS.

Channelrhodopsins, such as the algal phototaxis receptor Platymonas subcordiformis channelrhodopsin-2 (PsChR2), are light-gated cation channels used as optogenetic tools for photocontrol of membrane potential in living cells. Channelrhodopsin (ChR)-mediated photocurrent responses are complex and poorly understood, exhibiting alterations in peak current amplitude, extents and kinetics of inactivation, and kinetics of the recovery of the prestimulus dark current that are sensitive to duration and frequency of photostimuli. From the analysis of time-resolved optical absorption data, presented in the accompanying article, we derived a two-cycle model that describes the photocycles of PsChR2. Here, we applied the model to evaluate the transient currents produced by PsChR2 expressed in HEK293 cells under both fast laser excitation and step-like continuous illumination. Interpretation of the photocurrents in terms of the photocycle kinetics indicates that the O states in both cycles are responsible for the channel current and fit the current transients under the different illumination regimes. The peak and plateau currents in response to a single light step, a train of light pulses, and a light step superimposed on a continuous light background observed for ChR2 proteins are explained in terms of contributions from the two parallel photocycles. The analysis shows that the peak current desensitization and recovery phenomena are inherent properties of the photocycles. The light dependence of desensitization is reproduced and explained by the time evolution of the concentration transients in response to step-like illumination. Our data show that photocycle kinetic parameters are sufficient to explain the complex dependence of photocurrent responses to photostimuli.

Effects of macromolecular crowding on burst phase kinetics of cytochrome c folding.

Excluded volume and viscosity effects of crowding agents that mimic crowded conditions in vivo on "classical" burst phase folding kinetics of cytochrome c are assessed in vitro. Upon electron transfer-triggered folding of reduced cytochrome c, far-UV time-resolved circular dichroism (TRCD) is used to monitor folding under different conditions. Earlier work has shown that folding of reduced cytochrome c from the guanidinium hydrochloride-induced unfolded ensemble in dilute phosphate buffer involves kinetic partitioning: one fraction of molecules folds rapidly, on a time scale identical to that of reduction, while the remaining population folds more slowly. In the presence of 220 mg/mL dextran 70, a synthetic macromolecular crowding agent that occupies space but does not interact with proteins, the population of the fast folding step for cytochrome c is greatly reduced. Increasing the viscosity with sucrose to the same microviscosity exhibited by the dextran solution showed no significant decrease in the amplitude of the fast-folding phase of cytochrome c. Experiments show that the unfolded-state heme ligation remains bis-His in the presence of dextran 70, but coarse-grained simulations suggest that the unfolded-state ensemble becomes more compact in the presence of crowders. We conclude that excluded volume effects alter unfolded cytochrome c such that access to fast-folding conformations is reduced.

Deconstructing time-resolved optical rotatory dispersion kinetic measurements of cytochrome c folding: from molten globule to the native state.

The far-UV time-resolved optical rotatory dispersion (TRORD) technique has contributed significantly to our understanding of nanosecond secondary structure kinetics in protein folding and function reactions. For reduced cytochrome c, protein folding kinetics have been probed largely from the unfolded to the native state. Here we provide details about sample preparation and the TRORD apparatus and measurements for studying folding from a partly unfolded state to the native secondary structure conformation of reduced cytochrome c.