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1.
J Biol Chem ; 300(2): 105649, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38237683

RESUMO

Class A G protein-coupled receptors (GPCRs), a superfamily of cell membrane signaling receptors, moonlight as constitutively active phospholipid scramblases. The plasma membrane of metazoan cells is replete with GPCRs yet has a strong resting trans-bilayer phospholipid asymmetry, with the signaling lipid phosphatidylserine confined to the cytoplasmic leaflet. To account for the persistence of this lipid asymmetry in the presence of GPCR scramblases, we hypothesized that GPCR-mediated lipid scrambling is regulated by cholesterol, a major constituent of the plasma membrane. We now present a technique whereby synthetic vesicles reconstituted with GPCRs can be supplemented with cholesterol to a level similar to that of the plasma membrane and show that the scramblase activity of two prototypical GPCRs, opsin and the ß1-adrenergic receptor, is impaired upon cholesterol loading. Our data suggest that cholesterol acts as a switch, inhibiting scrambling above a receptor-specific threshold concentration to disable GPCR scramblases at the plasma membrane.


Assuntos
Fosfolipídeos , Receptores Acoplados a Proteínas G , Animais , Transporte Biológico , Colesterol , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Bovinos , Perus
2.
bioRxiv ; 2024 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-38045315

RESUMO

Class A G protein-coupled receptors (GPCRs), a superfamily of cell membrane signaling receptors, moonlight as constitutively active phospholipid scramblases. The plasma membrane of metazoan cells is replete with GPCRs, yet has a strong resting trans-bilayer phospholipid asymmetry, with the signaling lipid phosphatidylserine confined to the cytoplasmic leaflet. To account for the persistence of this lipid asymmetry in the presence of GPCR scramblases, we hypothesized that GPCR-mediated lipid scrambling is regulated by cholesterol, a major constituent of the plasma membrane. We now present a technique whereby synthetic vesicles reconstituted with GPCRs can be supplemented with cholesterol to a level similar to that of the plasma membrane and show that the scramblase activity of two prototypical GPCRs, opsin and the ß1-adrenergic receptor, is impaired upon cholesterol loading. Our data suggest that cholesterol acts as a switch, inhibiting scrambling above a receptor-specific threshold concentration to disable GPCR scramblases at the plasma membrane.

3.
PLoS Pathog ; 19(8): e1011532, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37531329

RESUMO

The COVID-19 pandemic represents a global challenge that has impacted and is expected to continue to impact the lives and health of people across the world for the foreseeable future. The rollout of vaccines has provided highly anticipated relief, but effective therapeutics are required to further reduce the risk and severity of infections. Monoclonal antibodies have been shown to be effective as therapeutics for SARS-CoV-2, but as new variants of concern (VoC) continue to emerge, their utility and use have waned due to limited or no efficacy against these variants. Furthermore, cumbersome systemic administration limits easy and broad access to such drugs. As well, concentrations of systemically administered antibodies in the mucosal epithelium, a primary site of initial infection, are dependent on neonatal Fc receptor mediated transport and require high drug concentrations. To reduce the viral load more effectively in the lung, we developed an inhalable formulation of a SARS-CoV-2 neutralizing antibody binding to a conserved epitope on the Spike protein, ensuring pan-neutralizing properties. Administration of this antibody via a vibrating mesh nebulization device retained antibody integrity and resulted in effective distribution of the antibody in the upper and lower respiratory tract of non-human primates (NHP). In comparison with intravenous administration, significantly higher antibody concentrations can be obtained in the lung, resulting in highly effective reduction in viral load post SARS-CoV-2 challenge. This approach may reduce the barriers of access and uptake of antibody therapeutics in real-world clinical settings and provide a more effective blueprint for targeting existing and potentially emerging respiratory tract viruses.


Assuntos
Antivirais , COVID-19 , Animais , Humanos , SARS-CoV-2 , Pandemias , Anticorpos Antivirais , Anticorpos Neutralizantes , Epitopos , Glicoproteína da Espícula de Coronavírus
4.
Nat Commun ; 14(1): 4365, 2023 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-37474513

RESUMO

Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is a light-gated channel used for optogenetic silencing of mammalian neurons. It selects K+ over Na+ in the absence of the canonical tetrameric K+ selectivity filter found universally in voltage- and ligand-gated channels. The genome of H. catenoides also encodes a highly homologous cation channelrhodopsin (HcCCR), a Na+ channel with >100-fold larger Na+ to K+ permeability ratio. Here, we use cryo-electron microscopy to determine atomic structures of these two channels embedded in peptidiscs to elucidate structural foundations of their dramatically different cation selectivity. Together with structure-guided mutagenesis, we show that K+ versus Na+ selectivity is determined at two distinct sites on the putative ion conduction pathway: in a patch of critical residues in the intracellular segment (Leu69/Phe69, Ile73/Ser73 and Asp116) and within a cluster of aromatic residues in the extracellular segment (primarily, Trp102 and Tyr222). The two filters are on the opposite sides of the photoactive site involved in channel gating.


Assuntos
Mamíferos , Animais , Channelrhodopsins/genética , Microscopia Crioeletrônica , Cátions/metabolismo , Mamíferos/metabolismo
5.
Sci Rep ; 12(1): 13955, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35977989

RESUMO

Within the microbial rhodopsin family, heliorhodopsins (HeRs) form a phylogenetically distinct group of light-harvesting retinal proteins with largely unknown functions. We have determined the 1.97 Å resolution X-ray crystal structure of Thermoplasmatales archaeon SG8-52-1 heliorhodopsin (TaHeR) in the presence of NaCl under acidic conditions (pH 4.5), which complements the known 2.4 Å TaHeR structure acquired at pH 8.0. The low pH structure revealed that the hydrophilic Schiff base cavity (SBC) accommodates a chloride anion to stabilize the protonated retinal Schiff base when its primary counterion (Glu-108) is neutralized. Comparison of the two structures at different pH revealed conformational changes connecting the SBC and the extracellular loop linking helices A-B. We corroborated this intramolecular signaling transduction pathway with computational studies, which revealed allosteric network changes propagating from the perturbed SBC to the intracellular and extracellular space, suggesting TaHeR may function as a sensory rhodopsin. This intramolecular signaling mechanism may be conserved among HeRs, as similar changes were observed for HeR 48C12 between its pH 8.8 and pH 4.3 structures. We additionally performed DEER experiments, which suggests that TaHeR forms possible dimer-of-dimer associations which may be integral to its putative functionality as a light sensor in binding a transducer protein.


Assuntos
Cloretos , Bases de Schiff , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Rodopsina/química , Rodopsinas Microbianas/química , Bases de Schiff/química , Transdução de Sinais
6.
J Am Chem Soc ; 142(40): 17057-17068, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-32915556

RESUMO

Site-specific modification of proteins with functional molecules provides powerful tools for researching and engineering proteins. Here we report a new chemical conjugation method which photocages highly reactive but chemically selective moieties, enabling the use of protein-inert amines for selective protein modification. New amino acids FnbY and FmnbY, bearing photocaged quinone methides (QMs), were genetically incorporated into proteins. Upon light activation, they generated highly reactive QM, which rapidly reacted with amine derivatives. This method features a rare combination of desired properties including fast kinetics, small and stable linkage, compatibility with low temperature, photocontrollability, and widely available reagents. Moreover, labeling via FnbY occurs on the ß-carbon, affording the shortest linkage to protein backbone which is essential for advanced studies involving orientation and distance. We installed various functionalities onto proteins and attached a spin label as close as possible to the protein backbone, achieving high resolution in double electron-electron paramagnetic resonance distance measurements.


Assuntos
Aminas/química , Indolquinonas/química , Proteínas/química , Coloração e Rotulagem/métodos , Aminoácidos/química , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Processos Fotoquímicos , Conformação Proteica , Processamento de Proteína Pós-Traducional , Solventes/química , Marcadores de Spin , Compostos de Sulfidrila/química , Temperatura
7.
J Biol Chem ; 295(44): 14793-14804, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-32703899

RESUMO

Microbial rhodopsins are versatile and ubiquitous retinal-binding proteins that function as light-driven ion pumps, light-gated ion channels, and photosensors, with potential utility as optogenetic tools for altering membrane potential in target cells. Insights from crystal structures have been central for understanding proton, sodium, and chloride transport mechanisms of microbial rhodopsins. Two of three known groups of anion pumps, the archaeal halorhodopsins (HRs) and bacterial chloride-pumping rhodopsins, have been structurally characterized. Here we report the structure of a representative of a recently discovered third group consisting of cyanobacterial chloride and sulfate ion-pumping rhodopsins, the Mastigocladopsis repens rhodopsin (MastR). Chloride-pumping MastR contains in its ion transport pathway a unique Thr-Ser-Asp (TSD) motif, which is involved in the binding of a chloride ion. The structure reveals that the chloride-binding mode is more similar to HRs than chloride-pumping rhodopsins, but the overall structure most closely resembles bacteriorhodopsin (BR), an archaeal proton pump. The MastR structure shows a trimer arrangement reminiscent of BR-like proton pumps and shows features at the extracellular side more similar to BR than the other chloride pumps. We further solved the structure of the MastR-T74D mutant, which contains a single amino acid replacement in the TSD motif. We provide insights into why this point mutation can convert the MastR chloride pump into a proton pump but cannot in HRs. Our study points at the importance of precise coordination and exact location of the water molecule in the active center of proton pumps, which serves as a bridge for the key proton transfer.


Assuntos
Cianobactérias/química , Mutação , Bombas de Próton/química , Rodopsinas Microbianas/química , Sítios de Ligação , Biopolímeros/química , Cristalografia por Raios X , Transporte de Íons , Conformação Proteica , Bombas de Próton/genética , Prótons , Retinaldeído/metabolismo , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/metabolismo
8.
J Phys Chem Lett ; 11(10): 3889-3896, 2020 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-32330041

RESUMO

Owing to the ultrafast time scale of the photoinduced reaction and high degree of spectral overlap among the reactant, product, and excited electronic states in bacteriorhodopsin (bR), it has been a challenge for traditional spectroscopies to resolve the interplay between vibrational dynamics and electronic processes occurring in the retinal chromophore of bR. Here, we employ ultrafast two-dimensional electronic photon echo spectroscopy to follow the early excited-state dynamics of bR preceding the isomerization. We detect an early periodic photoinduced absorptive signal that, employing a hybrid multiconfigurational quantum/molecular mechanical model of bR, we attribute to periodic mixing of the first and second electronic excited states (S1 and S2, respectively). This recurrent interaction between S1 and S2, induced by a bond length alternation of the retinal chromohore, supports the hypothesis that the ultrafast photoisomerization in bR is initiated by a process involving coupled nuclear and electronic motion on three different electronic states.


Assuntos
Bacteriorodopsinas/química , Teoria Quântica , Estrutura Molecular , Espectroscopia Fotoeletrônica
9.
J Biol Chem ; 294(39): 14215-14230, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31399513

RESUMO

Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein-coupled receptor (GPCR), can organize as rows of dimers. GPCR dimerization and oligomerization offer possibilities for allosteric regulation of GPCR activity, but the detailed structures and mechanism remain elusive. In this investigation, we made use of the high rhodopsin density in the native disc membranes and of a bifunctional cross-linker that preserves the native rhodopsin arrangement by covalently tethering rhodopsins via Lys residue side chains. We purified cross-linked rhodopsin dimers and reconstituted them into nanodiscs for cryo-EM analysis. We present cryo-EM structures of the cross-linked rhodopsin dimer as well as a rhodopsin dimer reconstituted into nanodiscs from purified monomers. We demonstrate the presence of a preferential 2-fold symmetrical dimerization interface mediated by transmembrane helix 1 and the cytoplasmic helix 8 of rhodopsin. We confirmed this dimer interface by double electron-electron resonance measurements of spin-labeled rhodopsin. We propose that this interface and the arrangement of two protomers is a prerequisite for the formation of the observed rows of dimers. We anticipate that the approach outlined here could be extended to other GPCRs or membrane receptors to better understand specific receptor dimerization mechanisms.


Assuntos
Nanopartículas/química , Multimerização Proteica , Rodopsina/química , Animais , Bovinos , Microscopia Crioeletrônica , Células HEK293 , Humanos , Domínios Proteicos , Rodopsina/ultraestrutura
10.
Sci Rep ; 9(1): 11283, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375689

RESUMO

Gloeobacter rhodopsin (GR) is a cyanobacterial proton pump which can be potentially applied to optogenetics. We solved the crystal structure of GR and found that it has overall similarity to the homologous proton pump from Salinibacter ruber, xanthorhodopsin (XR). We identified distinct structural characteristics of GR's hydrogen bonding network in the transmembrane domain as well as the displacement of extracellular sides of the transmembrane helices relative to those of XR. Employing Raman spectroscopy and flash-photolysis, we found that GR in the crystals exists in a state which displays retinal conformation and photochemical cycle similar to the functional form observed in lipids. Based on the crystal structure of GR, we selected a site for spin labeling to determine GR's oligomerization state using double electron-electron resonance (DEER) spectroscopy and demonstrated the pH-dependent pentamer formation of GR. Determination of the structure of GR as well as its pentamerizing propensity enabled us to reveal the role of structural motifs (extended helices, 3-omega motif and flipped B-C loop) commonly found among light-driven bacterial pumps in oligomer formation. Here we propose a new concept to classify these pumps based on the relationship between their oligomerization propensities and these structural determinants.


Assuntos
Bacteroidetes/ultraestrutura , Conformação Proteica , Bombas de Próton/ultraestrutura , Rodopsina/ultraestrutura , Sequência de Aminoácidos/genética , Proteínas de Bactérias/ultraestrutura , Bacteroidetes/química , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Ligação de Hidrogênio , Multimerização Proteica/genética , Bombas de Próton/síntese química , Bombas de Próton/química , Rodopsina/química , Rodopsina/genética , Rodopsinas Microbianas/ultraestrutura , Análise Espectral Raman
11.
Nature ; 561(7724): E44, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29930353

RESUMO

In the PDF version of this Article, owing to a typesetting error, an incorrect figure was used for Extended Data Fig. 5; the correct figure was used in the HTML version. This has been corrected online.

12.
Nature ; 558(7711): 553-558, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29899450

RESUMO

G-protein-coupled receptors comprise the largest family of mammalian transmembrane receptors. They mediate numerous cellular pathways by coupling with downstream signalling transducers, including the hetrotrimeric G proteins Gs (stimulatory) and Gi (inhibitory) and several arrestin proteins. The structural mechanisms that define how G-protein-coupled receptors selectively couple to a specific type of G protein or arrestin remain unknown. Here, using cryo-electron microscopy, we show that the major interactions between activated rhodopsin and Gi are mediated by the C-terminal helix of the Gi α-subunit, which is wedged into the cytoplasmic cavity of the transmembrane helix bundle and directly contacts the amino terminus of helix 8 of rhodopsin. Structural comparisons of inactive, Gi-bound and arrestin-bound forms of rhodopsin with inactive and Gs-bound forms of the ß2-adrenergic receptor provide a foundation to understand the unique structural signatures that are associated with the recognition of Gs, Gi and arrestin by activated G-protein-coupled receptors.


Assuntos
Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Rodopsina/metabolismo , Rodopsina/ultraestrutura , Arrestina/química , Arrestina/metabolismo , Sítios de Ligação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Modelos Moleculares , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Rodopsina/química , Transdução de Sinais , Especificidade por Substrato
13.
Nat Protoc ; 13(2): 260-292, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29300389

RESUMO

Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20-25°C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basic screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in ≤90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min.


Assuntos
Cristalografia por Raios X/métodos , Ensaios de Triagem em Larga Escala/métodos , Cristalização , Coleta de Dados , Ensaios de Triagem em Larga Escala/instrumentação , Lipídeos , Proteínas de Membrana/análise , Polietilenotereftalatos/química , Proteínas/química , Temperatura , Raios X
14.
Sci Rep ; 7(1): 9522, 2017 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-28842688

RESUMO

The retinylidene protein bacteriorhodopsin (BR) is a heptahelical light-dependent proton pump found in the purple membrane of the archaeon Halobacterium salinarum. We now show that when reconstituted into large unilamellar vesicles, purified BR trimers exhibit light-independent lipid scramblase activity, thereby facilitating transbilayer exchange of phospholipids between the leaflets of the vesicle membrane at a rate >10,000 per trimer per second. This activity is comparable to that of recently described scramblases including bovine rhodopsin and fungal TMEM16 proteins. Specificity tests reveal that BR scrambles fluorescent analogues of common phospholipids but does not transport a glycosylated diphosphate isoprenoid lipid. In silico analyses suggest that membrane-exposed polar residues in transmembrane helices 1 and 2 of BR may provide the molecular basis for lipid translocation by coordinating the polar head-groups of transiting phospholipids. Consistent with this possibility, extensive coarse-grained molecular dynamics simulations of a BR trimer in an explicit phospholipid membrane revealed water penetration along transmembrane helix 1 with the cooperation of a polar residue (Y147 in transmembrane helix 5) in the adjacent protomer. These results suggest that the lipid translocation pathway may lie at or near the interface of the protomers of a BR trimer.


Assuntos
Bacteriorodopsinas/metabolismo , Halobacterium salinarum/metabolismo , Halobacterium salinarum/efeitos da radiação , Luz , Proteínas de Transferência de Fosfolipídeos/metabolismo , Bacteriorodopsinas/química , Modelos Moleculares , Proteínas de Transferência de Fosfolipídeos/química , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Conformação Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade
15.
Proc Natl Acad Sci U S A ; 114(16): E3268-E3275, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28373559

RESUMO

Conformational equilibria of G-protein-coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron-electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ∼16 min at 20 °C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners.


Assuntos
Luz , Nanoestruturas/química , Conformação Proteica/efeitos da radiação , Rodopsina/química , Transducina/química , Animais , Bovinos , Estrutura Secundária de Proteína , Rodopsina/metabolismo , Rodopsina/efeitos da radiação , Marcadores de Spin , Transducina/metabolismo , Transducina/efeitos da radiação
16.
J Phys Chem B ; 121(16): 4040-4047, 2017 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-28358485

RESUMO

Ultrafast photochemical reactions are initiated by vibronic transitions from the reactant ground state to the excited potential energy surface, directly populating excited-state vibrational modes. The primary photochemical reaction of vision, the isomerization of retinal in the protein rhodopsin, is known to be a vibrationally coherent reaction, but the Franck-Condon factors responsible for initiating the process have been difficult to resolve with conventional time-resolved spectroscopies. Here we employ experimental and theoretical 2D photon echo spectroscopy to directly resolve for the first time the Franck-Condon factors that initiate isomerization on the excited potential energy surface and track the reaction dynamics. The spectral dynamics reveal vibrationally coherent isomerization occurring on the fastest possible time scale, that of a single period of the local torsional reaction coordinate. We successfully model this process as coherent wavepacket motion through a conical intersection on a ∼30 fs time scale, confirming the reaction coordinate as a local torsional coordinate with a frequency of ∼570 cm-1. As a result of spectral features being spread out along two frequency coordinates, we unambiguously assign reactant and product states following passage through the conical intersection, which reveal the key vibronic transitions that initiate the vibrationally coherent photochemistry of vision.

17.
Nat Commun ; 7: 12832, 2016 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-27694816

RESUMO

Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsin's activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease. Here we show that three such enigmatic mutations-F45L, V209M and F220C-yield fully functional visual pigments that bind the 11-cis retinal chromophore, activate the G protein transducin, traffic to the light-sensitive photoreceptor compartment and scramble phospholipids. However, tests of scramblase activity show that unlike wild-type rhodopsin that functionally reconstitutes into liposomes as dimers or multimers, F45L, V209M and F220C rhodopsins behave as monomers. This result was confirmed in pull-down experiments. Our data suggest that the photoreceptor pathology associated with expression of these enigmatic RP-associated pigments arises from their unexpected inability to dimerize via transmembrane helices 1 and 5.


Assuntos
Mutação , Mutação Puntual , Retina/metabolismo , Retinose Pigmentar/genética , Rodopsina/química , Rodopsina/genética , Animais , Células COS , Bovinos , Chlorocebus aethiops , Proteínas de Ligação ao GTP/química , Células HEK293 , Humanos , Lipossomos/metabolismo , Camundongos Knockout , Proteínas de Transferência de Fosfolipídeos/metabolismo , Multimerização Proteica , Retina/química , Transducina/genética
18.
Nat Chem ; 7(12): 980-6, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26587713

RESUMO

The role of vibrational coherence-concerted vibrational motion on the excited-state potential energy surface-in the isomerization of retinal in the protein rhodopsin remains elusive, despite considerable experimental and theoretical efforts. We revisited this problem with resonant ultrafast heterodyne-detected transient-grating spectroscopy. The enhanced sensitivity that this technique provides allows us to probe directly the primary photochemical reaction of vision with sufficient temporal and spectral resolution to resolve all the relevant nuclear dynamics of the retinal chromophore during isomerization. We observed coherent photoproduct formation on a sub-50 fs timescale, and recovered a host of vibrational modes of the retinal chromophore that modulate the transient-grating signal during the isomerization reaction. Through Fourier filtering and subsequent time-domain analysis of the transient vibrational dynamics, the excited-state nuclear motions that drive the isomerization reaction were identified, and comprise stretching, torsional and out-of-plane wagging motions about the local C11=C12 isomerization coordinate.


Assuntos
Rodopsina/química , Vibração , Animais , Bovinos , Isomerismo , Fotoquímica , Análise Espectral
19.
Photochem Photobiol Sci ; 14(9): 1586-97, 2015 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-26140679

RESUMO

Electron paramagnetic resonance (EPR) spectroscopy, together with spin labeling techniques, has played a major role in the characterization of rhodopsin, the photoreceptor protein and G protein-coupled receptor (GPCR) in rod cells. Two decades ago, these biophysical tools were the first to identify transmembrane helical movements in rhodopsin upon photo-activation, a critical step in the study of GPCR signaling. EPR methods were employed to identify functional loop dynamics within rhodopsin, to measure light-induced millisecond timescale changes in rhodopsin conformation, to characterize the effects of partial agonists on the apoprotein opsin, and to study lipid interactions with rhodopsin. With the emergence of advanced pulsed EPR techniques, the stage was set to determine the amplitude of structural changes in rhodopsin and the dynamics in the rhodopsin signaling complexes. Work in this area has yielded invaluable information about mechanistic properties of GPCRs. Using EPR techniques, receptors are studied in native-like membrane environments and the effects of lipids on conformational equilibria can be explored. This perspective addresses the impact of EPR methods on rhodopsin and GPCR structural biology, highlighting historical discoveries made with spin labeling techniques, and outlining exciting new directions in the field.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Rodopsina/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Humanos , Rodopsina/química
20.
Nat Commun ; 5: 5115, 2014 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-25296113

RESUMO

Opsin, the rhodopsin apoprotein, was recently shown to be an ATP-independent flippase (or scramblase) that equilibrates phospholipids across photoreceptor disc membranes in mammalian retina, a process required for disc homoeostasis. Here we show that scrambling is a constitutive activity of rhodopsin, distinct from its light-sensing function. Upon reconstitution into vesicles, discrete conformational states of the protein (rhodopsin, a metarhodopsin II-mimic, and two forms of opsin) facilitated rapid (>10,000 phospholipids per protein per second) scrambling of phospholipid probes. Our results indicate that the large conformational changes involved in converting rhodopsin to metarhodopsin II are not required for scrambling, and that the lipid translocation pathway either lies near the protein surface or involves membrane packing defects in the vicinity of the protein. In addition, we demonstrate that ß2-adrenergic and adenosine A2A receptors scramble lipids, suggesting that rhodopsin-like G protein-coupled receptors may play an unexpected moonlighting role in re-modelling cell membranes.


Assuntos
Proteínas de Transferência de Fosfolipídeos/fisiologia , Receptores Adrenérgicos beta 2/fisiologia , Rodopsina/fisiologia , Animais , Bovinos , Opsinas/fisiologia , Conformação Proteica , Receptores A2 de Adenosina/fisiologia
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