RESUMO
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.
Assuntos
Optogenética , Channelrhodopsins/metabolismo , Ânions , Cinética , Optogenética/métodosRESUMO
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.
Assuntos
Ativação do Canal Iônico , Cinética , Rodopsina/metabolismo , Rodopsina/química , Rodopsina/genética , Animais , Ânions/metabolismo , Luz , Modelos Biológicos , Channelrhodopsins/metabolismo , Channelrhodopsins/genética , Channelrhodopsins/químicaRESUMO
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.
Assuntos
Criptófitas , Optogenética , Channelrhodopsins/metabolismo , Ânions/metabolismo , Criptófitas/metabolismo , Optogenética/métodos , LuzRESUMO
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.
Assuntos
Nanopartículas , Rodopsina , Cinética , Bicamadas Lipídicas , Lipídeos , Maleatos , PoliestirenosRESUMO
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.
Assuntos
Proteínas de Membrana , Rodopsina , Lipídeos , MaleatosRESUMO
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.
Assuntos
Bicamadas Lipídicas , Rodopsina , Eletrônica , Lipídeos de Membrana , Fosfatidilcolinas , Análise EspectralRESUMO
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.
Assuntos
Proteínas Luminescentes/química , Multimerização Proteica , Temperatura , Células HEK293 , Humanos , Modelos Moleculares , Estrutura Quaternária de ProteínaRESUMO
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 â¼7% 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.
Assuntos
Lipídeos de Membrana/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Humanos , CinéticaRESUMO
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.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Dicroísmo Circular , Humanos , Mutação , Estrutura Secundária de Proteína , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Álcalis/química , Temperatura Baixa , Rodopsina/metabolismo , Animais , Bovinos , Concentração de Íons de Hidrogênio , Análise Espectral/métodosRESUMO
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.
Assuntos
Luz , Fotólise , Rodopsina/química , Bases de Schiff/química , Animais , Bovinos , Humanos , CinéticaRESUMO
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 (â¼60%) and ß (â¼10%) chains, resulting in a modest barrier to ligand binding. A subsequent phase, concurrent with the first step of the R â 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 (â¼90%). 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.
Assuntos
Hemoglobinas/química , Regulação Alostérica , Heme/química , Humanos , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Fotólise , Estrutura Quaternária de Proteína , Água/química , alfa-Globinas/química , Globinas beta/químicaRESUMO
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.
Assuntos
Clorófitas/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Clorófitas/química , Clorófitas/genética , Clorófitas/efeitos da radiação , Células HEK293 , Humanos , Cinética , Luz , Rodopsina/genéticaRESUMO
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.
Assuntos
Clorófitas/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Clorófitas/química , Clorófitas/genética , Clorófitas/efeitos da radiação , Cinética , Luz , Fotoquímica , Rodopsina/genéticaRESUMO
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.
Assuntos
Citocromos c/química , Substâncias Macromoleculares , Dicroísmo Circular , Cinética , Dobramento de Proteína , Espectrofotometria UltravioletaRESUMO
Molecular structure and function studies of vertebrate ultraviolet (UV) cone visual pigments are needed to understand the molecular evolution of these photoreceptors, which uniquely contain unprotonated Schiff base linkages between the 11-cis-retinal chromophore and the opsin proteins. In this study, the Siberian hamster ultraviolet cone pigment (SHUV) was expressed and purified in an n-dodecyl-ß-D-maltoside suspension for optical characterization. Time-resolved absorbance measurements, over a spectral range from 300 to 700 nm, were taken for the purified pigment at time delays from 30 ns to 4.64 s after photoexcitation using 7 ns pulses of 355 nm light. The resulting data were fit globally to a sum of exponential functions after noise reduction using singular-value decomposition. Four exponentials best fit the data with lifetimes of 1.4 µs, 210 µs, 47 ms, and 1 s. The first photointermediate species characterized here is an equilibrated mixture similar to the one formed after rhodopsin's Batho intermediate decays into equilibrium with its successor, BSI. The extremely large red shift of the SHUV Batho component relative to the pigment suggests that SHUV Batho has a protonated Schiff base and that the SHUV cone pigment itself has an unprotonated Schiff base. In contrast to SHUV Batho, the portion of the equilibrated mixture's spectrum corresponding to SHUV BSI is well fit by a model spectrum with an unprotonated Schiff base. The spectra of the next two photointermediate species revealed that they both have unprotonated Schiff bases and suggest they are analogous to rhodopsin's Lumi I and Lumi II species. After decay of SHUV Lumi II, the correspondence with rhodopsin photointermediates breaks down and the next photointermediate, presumably including the G protein-activating species, is a mixture of protonated and unprotonated Schiff base photointermediate species.
Assuntos
Fotólise , Prótons , Células Fotorreceptoras Retinianas Cones/metabolismo , Pigmentos da Retina/química , Bases de Schiff/química , Raios Ultravioleta , Animais , Cricetinae , Células HEK293 , Humanos , Phodopus , Células Fotorreceptoras Retinianas Cones/efeitos da radiação , Pigmentos da Retina/metabolismoRESUMO
Recent and ongoing developments in time-resolved spectroscopy have made it possible to monitor circular dichroism, magnetic circular dichroism, optical rotatory dispersion, and magnetic optical rotatory dispersion with nanosecond time resolution. These techniques have been applied to determine structural changes associated with the function of several proteins as well as to determine the nature of early events in protein folding. These studies have required new approaches in triggering protein reactions as well as the development of time-resolved techniques for polarization spectroscopies with sufficient time resolution and sensitivity to probe protein structural changes.
Assuntos
Proteínas/metabolismo , Monóxido de Carbono/química , Dicroísmo Circular , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Cinética , Magnetismo , Dispersão Óptica Rotatória , Fotólise , Dobramento de Proteína , Proteínas/químicaRESUMO
Time-dependent studies of membrane protein function are hindered by extensive light scattering that impedes application of fast optical absorbance methods. Detergent solubilization reduces light scattering but strongly perturbs rhodopsin activation kinetics. Nanodiscs may be a better alternative if they can be shown to be free from the serious kinetic perturbations associated with detergent solubilization. To resolve this, we monitored absorbance changes due to photointermediates formed on the microsecond to hundred millisecond time scale after excitation of bovine rhodopsin nanodiscs and compared them to photointermediates that form in hypotonically washed native membranes as well as to those that form in lauryl maltoside suspensions at 15 and 30 °C over a pH range from 6.5 to 8.7. Time-resolved difference spectra were collected from 300 to 700 nm at a series of time delays after photoexcitation and globally fit to a sum of time-decaying exponential terms, and the photointermediates present were determined from the spectral coefficients of the exponential terms. At the temperatures and pHs studied, photointermediates formed after photoexcitation of rhodopsin in nanodiscs are extremely similar to those that form in native membrane, in particular displaying the normal forward shift of the Meta I(480) â Meta II equilibrium with increased temperature and reduced pH which occurs in native membrane but which is not observed in lauryl maltoside detergent suspensions. These results were obtained using the amount of rhodopsin in nanodiscs which is required for optical experiments with rhodopsin mutants. This work demonstrates that late, physiologically important rhodopsin photointermediates can be characterized in nanodiscs, which provide the superior optical properties of detergent without perturbing the activation sequence.
Assuntos
Nanoestruturas/química , Rodopsina/química , Animais , Bovinos , Glucosídeos/química , Glucosídeos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete , TemperaturaRESUMO
Polarization methods, introduced in the 1800s, offered one of the earliest ways to examine protein structure. Since then, many other structure-sensitive probes have been developed, but circular dichroism (CD) remains a powerful technique because of its versatility and the specificity of protein structural information that can be explored. With improvements in time resolution, from millisecond to picosecond CD measurements, it has proven to be an important tool for studying the mechanism of folding and function in many biomolecules. For example, nanosecond time-resolved CD (TRCD) studies of the sub-microsecond events of reduced cytochrome c folding have provided direct experimental evidence of kinetic heterogeneity, which is an inherent property of the diffusional nature of early folding dynamics on the energy landscape. In addition, TRCD has been applied to the study of many biochemical processes, such as ligand rebinding in hemoglobin and myoglobin and signaling state formation in photoactive yellow protein and prototropin 1 LOV2. The basic approach to TRCD has also been extended to include a repertoire of nanosecond polarization spectroscopies: optical rotatory dispersion (ORD), magnetic CD and ORD, and linear dichroism. This article will discuss the details of the polarization methods used in this laboratory, as well as the coupling of time-resolved ORD with the temperature-jump trigger so that protein folding can be studied in a larger number of proteins.
Assuntos
Dobramento de Proteína , Proteínas/química , Dicroísmo Circular , Cinética , Estrutura Secundária de Proteína , Análise Espectral , Análise Espectral RamanRESUMO
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.