RESUMO
Arrestins are a small family of proteins that bind G protein-coupled receptors (GPCRs). Arrestin binds to active phosphorylated GPCRs with higher affinity than to all other functional forms of the receptor, including inactive phosphorylated and active unphosphorylated. The selectivity of arrestins suggests that they must have two sensors, which detect receptor-attached phosphates and the active receptor conformation independently. Simultaneous engagement of both sensors enables arrestin transition into a high-affinity receptor-binding state. This transition involves a global conformational rearrangement that brings additional elements of the arrestin molecule, including the middle loop, in contact with a GPCR, thereby stabilizing the complex. Here, we review structural and mutagenesis data that identify these two sensors and additional receptor-binding elements within the arrestin molecule. While most data were obtained with the arrestin-1-rhodopsin pair, the evidence suggests that all arrestins use similar mechanisms to achieve preferential binding to active phosphorylated GPCRs.
Assuntos
Arrestina/ultraestrutura , Receptores Acoplados a Proteínas G/ultraestrutura , Rodopsina/ultraestrutura , Arrestina/genética , Sítios de Ligação/genética , Humanos , Mutagênese/genética , Fosforilação , Ligação Proteica/genética , Conformação Proteica , Receptores Acoplados a Proteínas G/genética , Rodopsina/genéticaRESUMO
G-protein coupled receptors (GPCRs) are known to form homo- and hetero- oligomers which are considered critical to modulate their function. However, studying the existence and functional implication of these complexes is not straightforward as controversial results are obtained depending on the method of analysis employed. Here, we use a quantitative single molecule super-resolution imaging technique named qPAINT to quantify complex formation within an example GPCR. qPAINT, based upon DNA-PAINT, takes advantage of the binding kinetics between fluorescently labelled DNA imager strands to complementary DNA docking strands coupled to protein targeting antibodies to quantify the protein copy number in nanoscale dimensions. We demonstrate qPAINT analysis via a novel pipeline to study the oligomerization of the purinergic receptor Y2 (P2Y2), a rhodopsin-like GPCR, highly expressed in the pancreatic cancer cell line AsPC-1, under control, agonistic and antagonistic conditions. Results reveal that whilst the density of P2Y2 receptors remained unchanged, antagonistic conditions displayed reduced percentage of oligomers, and smaller numbers of receptors in complexes. Yet, the oligomeric state of the receptors was not affected by agonist treatment, in line with previous reports. Understanding P2Y2 oligomerization under agonistic and antagonistic conditions will contribute to unravelling P2Y2 mechanistic action and therapeutic targeting.
Assuntos
Neoplasias Pancreáticas/genética , Multimerização Proteica/genética , Receptores Acoplados a Proteínas G/genética , Receptores Purinérgicos P2Y2/genética , DNA/genética , Humanos , Cinética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptores Acoplados a Proteínas G/ultraestrutura , Receptores Purinérgicos P2Y2/ultraestrutura , Rodopsina/genética , Rodopsina/ultraestrutura , Transdução de Sinais/genéticaRESUMO
Rhodopsin (RHO) misfolding mutations are a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). The most prevalent mutation, RHOP23H, results in its misfolding and retention in the endoplasmic reticulum (ER). Under homeostatic conditions, misfolded proteins are selectively identified, retained at the ER, and cleared via ER-associated degradation (ERAD). Overload of these degradation processes for a prolonged period leads to imbalanced proteostasis and may eventually result in cell death. ERAD of misfolded proteins, such as RHOP23H, includes the subsequent steps of protein recognition, targeting for ERAD, retrotranslocation, and proteasomal degradation. In the present study, we investigated and compared pharmacological modulation of ERAD at these four different major steps. We show that inhibition of the VCP/proteasome activity favors cell survival and suppresses P23H-mediated retinal degeneration in RHOP23H rat retinal explants. We suggest targeting this activity as a therapeutic approach for patients with currently untreatable adRP.
Assuntos
Retículo Endoplasmático/efeitos dos fármacos , Degeneração Retiniana/genética , Retinose Pigmentar/genética , Rodopsina/genética , Alcaloides/farmacologia , Animais , Animais Geneticamente Modificados , Benzoquinonas/farmacologia , Modelos Animais de Doenças , Retículo Endoplasmático/genética , Humanos , Lactamas Macrocíclicas/farmacologia , Mutação/genética , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética , Dobramento de Proteína/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Ratos , Retina/efeitos dos fármacos , Retina/crescimento & desenvolvimento , Retina/patologia , Degeneração Retiniana/patologia , Retinose Pigmentar/patologia , Rodopsina/ultraestruturaRESUMO
Channelrhodopsins (ChR) are light-gated ion-channels heavily used in optogenetics. Upon light excitation an ultrafast all-trans to 13-cis isomerization of the retinal chromophore takes place. It is still uncertain by what means this reaction leads to further protein changes and channel conductivity. Channelrhodopsin-1 in Chlamydomonas augustae exhibits a 100 fs photoisomerization and a protonated counterion complex. By polarization resolved ultrafast spectroscopy in the mid-IR we show that the initial reaction of the retinal is accompanied by changes in the protein backbone and ultrafast protonation changes at the counterion complex comprising Asp299 and Glu169. In combination with homology modelling and quantum mechanics/molecular mechanics (QM/MM) geometry optimization we assign the protonation dynamics to ultrafast deprotonation of Glu169, and transient protonation of the Glu169 backbone, followed by a proton transfer from the backbone to the carboxylate group of Asp299 on a timescale of tens of picoseconds. The second proton transfer is not related to retinal dynamics and reflects pure protein changes in the first photoproduct. We assume these protein dynamics to be the first steps in a cascade of protein-wide changes resulting in channel conductivity.
Assuntos
Channelrhodopsins/química , Modelos Moleculares , Conformação Proteica , Rodopsina/química , Channelrhodopsins/ultraestrutura , Chlamydomonas/química , Isomerismo , Luz , Simulação de Dinâmica Molecular , Prótons , Teoria Quântica , Rodopsina/ultraestrutura , Análise EspectralRESUMO
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/ultraestruturaRESUMO
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 RamanRESUMO
One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently heterotrimeric G proteins. The recent surge in structural data has expanded our understanding of GPCR-mediated signal transduction. However, many aspects, including the existence of transient interactions, remain elusive. We present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. Our density map reveals the receptor C-terminal tail bound to the Gß subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of Gß as scaffold for recruiting Gα subunits and G protein-receptor kinases. By comparing available complexes, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/ultraestrutura , Subunidades beta da Proteína de Ligação ao GTP/ultraestrutura , Subunidades gama da Proteína de Ligação ao GTP/ultraestrutura , Rodopsina/ultraestrutura , Animais , Bovinos , Microscopia Crioeletrônica , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Rodopsina/metabolismoRESUMO
Light-sensitive G protein-coupled receptors (GPCRs)-rhodopsins-absorb photons to isomerize their covalently bound retinal, triggering conformational changes that result in downstream signaling cascades. Monostable rhodopsins release retinal upon isomerization as opposed to the retinal in bistable rhodopsins that "reisomerize" upon absorption of a second photon. Understanding the mechanistic differences between these light-sensitive GPCRs has been hindered by the scarcity of recombinant models of the latter. Here, we reveal the high-resolution crystal structure of a recombinant bistable rhodopsin, jumping spider rhodopsin-1, bound to the inverse agonist 9-cis retinal. We observe a water-mediated network around the ligand hinting toward the basis of their bistable nature. In contrast to bovine rhodopsin (monostable), the transmembrane bundle of jumping spider rhodopsin-1 as well that of the bistable squid rhodopsin adopts a more "activation-ready" conformation often observed in other nonphotosensitive class A GPCRs. These similarities suggest the role of jumping spider rhodopsin-1 as a potential model system in the study of the structure-function relationship of both photosensitive and nonphotosensitive class A GPCRs.
Assuntos
Proteínas de Artrópodes/ultraestrutura , Rodopsina/ultraestrutura , Transdução de Sinais/efeitos da radiação , Aranhas , Animais , Proteínas de Artrópodes/isolamento & purificação , Proteínas de Artrópodes/metabolismo , Cristalografia por Raios X , Células HEK293 , Humanos , Ligantes , Luz , Simulação de Dinâmica Molecular , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Rodopsina/isolamento & purificação , Rodopsina/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The supramolecular organization of the visual pigment rhodopsin in the photoreceptor membrane remains contentious. Specifically, whether this G protein-coupled receptor functions as a monomer or dimer remains unknown, as does the presence or absence of ordered packing of rhodopsin molecules in the photoreceptor membrane. Completely opposite opinions have been expressed on both issues. Herein, using small-angle neutron and X-ray scattering approaches, we performed a comparative analysis of the structural characteristics of the photoreceptor membrane samples in buffer, both in the outer segment of photoreceptor cells, and in the free photoreceptor disks. The average distance between the centers of two neighboring rhodopsin molecules was found to be ~5.8â¯nm in both cases. The results indicate an unusually high packing density of rhodopsin molecules in the photoreceptor membrane, but molecules appear to be randomly distributed in the membrane without any regular ordering.
Assuntos
Células Fotorreceptoras/química , Células Fotorreceptoras/fisiologia , Rodopsina/química , Animais , Bovinos , Membrana Celular/química , Membranas , Difração de Nêutrons/métodos , Nêutrons , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/ultraestrutura , Retina/metabolismo , Rodopsina/metabolismo , Rodopsina/ultraestrutura , Espalhamento a Baixo ÂnguloRESUMO
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 SubstratoRESUMO
There is growing interest in studying and engineering integral membrane proteins (MPs) that play key roles in sensing and regulating cellular response to diverse external signals. A MP must be expressed, correctly inserted and folded in a lipid bilayer, and trafficked to the proper cellular location in order to function. The sequence and structural determinants of these processes are complex and highly constrained. Here we describe a predictive, machine-learning approach that captures this complexity to facilitate successful MP engineering and design. Machine learning on carefully-chosen training sequences made by structure-guided SCHEMA recombination has enabled us to accurately predict the rare sequences in a diverse library of channelrhodopsins (ChRs) that express and localize to the plasma membrane of mammalian cells. These light-gated channel proteins of microbial origin are of interest for neuroscience applications, where expression and localization to the plasma membrane is a prerequisite for function. We trained Gaussian process (GP) classification and regression models with expression and localization data from 218 ChR chimeras chosen from a 118,098-variant library designed by SCHEMA recombination of three parent ChRs. We use these GP models to identify ChRs that express and localize well and show that our models can elucidate sequence and structure elements important for these processes. We also used the predictive models to convert a naturally occurring ChR incapable of mammalian localization into one that localizes well.
Assuntos
Membrana Celular/química , Desenho de Fármacos , Canais Iônicos/química , Bicamadas Lipídicas/química , Aprendizado de Máquina , Rodopsina/química , Análise de Sequência de Proteína/métodos , Membrana Celular/ultraestrutura , Células HEK293 , Humanos , Canais Iônicos/ultraestrutura , Rodopsina/ultraestrutura , Relação Estrutura-Atividade , Frações Subcelulares/químicaRESUMO
A collection of crystal structures of rhodopsin, ß2-adrenergic and adenosine A2A receptors in active, intermediate and inactive states were selected for structural and energetic analyses to identify the changes involved in the activation/deactivation of Class A GPCRs. A set of helix interactions exclusive to either inactive or active/intermediate states were identified. The analysis of these interactions distinguished some local conformational changes involved in receptor activation, in particular, a packing between the intracellular domains of transmembrane helices H3 and H7 and a separation between those of H2 and H6. Also, differential movements of the extracellular and intracellular domains of these helices are apparent. Moreover, a segment of residues in helix H3, including residues L/I3.40 to L3.43, is identified as a key component of the activation mechanism, acting as a conformational hinge between extracellular and intracellular regions. Remarkably, the influence on the activation process of some glutamic and aspartic acidic residues and, as a consequence, the influence of variations on local pH is highlighted. Structural hypotheses that arose from the analysis of rhodopsin, ß2-adrenergic and adenosine A2A receptors were tested on the active and inactive M2 muscarinic acetylcholine receptor structures and further discussed in the context of the new mechanistic insights provided by the recently determined active and inactive crystal structures of the µ-opioid receptor. Overall, the structural and energetic analyses of the interhelical interactions present in this collection of Class A GPCRs suggests the existence of a common general activation mechanism featuring a chemical space useful for drug discovery exploration.
Assuntos
Receptor A2A de Adenosina/ultraestrutura , Receptor Muscarínico M2/ultraestrutura , Receptores Adrenérgicos beta 2/ultraestrutura , Receptores Opioides mu/ultraestrutura , Rodopsina/ultraestrutura , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática/fisiologia , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Receptor A2A de Adenosina/metabolismo , Receptor Muscarínico M2/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Opioides mu/metabolismo , Rodopsina/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 Å and 2.2 and 2.8 Å, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics.
Assuntos
Flavobacteriaceae/enzimologia , Rodopsina/ultraestrutura , ATPase Trocadora de Sódio-Potássio/ultraestrutura , Cristalografia por Raios X , Transporte de Íons , Modelos Moleculares , Potássio/metabolismo , Estrutura Terciária de Proteína , Sódio/metabolismoRESUMO
G protein-coupled receptors (GPCRs) span cell membranes with seven transmembrane helices and respond to a diverse array of extracellular signals. Crystal structures of GPCRs have provided key insights into the architecture of these receptors and the role of conserved residues. However, the question of how ligand binding induces the conformational changes that are essential for activation remains largely unanswered. Since the extracellular sequences and structures of GPCRs are not conserved between receptor subfamilies, it is likely that the initial molecular triggers for activation vary depending on the specific type of ligand and receptor. In this article, we describe NMR studies on the rhodopsin subfamily of GPCRs and propose a mechanism for how retinal isomerization switches the receptor to the active conformation. These results suggest a general approach for determining the triggers for activation in other GPCR subfamilies using NMR spectroscopy.
Assuntos
Aminoácidos/química , Ressonância Magnética Nuclear Biomolecular/métodos , Rodopsina/química , Rodopsina/ultraestrutura , Sítios de Ligação , Ligação Proteica , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/ultraestrutura , Relação Estrutura-AtividadeRESUMO
After 25 years of intensive research, the understanding of how photoreceptors in the eye perceive light and convert it into nerve signals has largely advanced. Central to this is the structural and mechanistic exploration of the G protein-coupled receptor rhodopsin acting as a dim-light sensing pigment in the retina. Investigation of rhodopsin by X-ray crystallographic, electron microscopic, and biochemical means depends on the ability to produce and isolate pure rhodopsin protein. Robust and well-defined protocols permit the production and crystallization of rhodopsin variants to investigate the inactive ground, the fully activated metarhodopsin II state, or disease-causing rhodopsin mutations. This chapter details how we express and purify biologically active variants of rhodopsin from HEK293S GnTI(-) cells in a quality and quantity suitable for biochemical assays, crystallization, and structure determination.
Assuntos
Rodopsina/química , Linhagem Celular , Cristalografia por Raios X , Humanos , Microscopia Eletrônica , Retina/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Rodopsina/ultraestruturaRESUMO
Inherently unstable, detergent-solubilized membrane protein complexes can often not be crystallized. For complexes that have a mass of >300 kDa, cryo-electron microscopy (EM) allows their three-dimensional (3D) structure to be assessed to a resolution that makes secondary structure elements visible in the best case. However, many interesting complexes exist whose mass is below 300 kDa and thus need alternative approaches. Two methods are reviewed: (i) Mass measurement in a scanning transmission electron microscope, which has provided important information on the stoichiometry of membrane protein complexes. This technique is applicable to particulate, filamentous and sheet-like structures. (ii) 3D-EM of negatively stained samples, which determines the molecular envelope of small membrane protein complexes. Staining and dehydration artifacts may corrupt the quality of the 3D map. Staining conditions thus need to be optimized. 3D maps of plant aquaporin SoPIP2;1 tetramers solubilized in different detergents illustrate that the flattening artifact can be partially prevented and that the detergent itself contributes significantly. Another example discussed is the complex of G protein-coupled receptor rhodopsin with its cognate G protein transducin.
Assuntos
Regulação Fúngica da Expressão Gênica , Imageamento Tridimensional/métodos , Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Pichia/genética , Aquaporinas/química , Aquaporinas/ultraestrutura , Microscopia Crioeletrônica/métodos , Detergentes/análise , Estudos de Avaliação como Assunto , Proteínas de Membrana/química , Coloração Negativa/métodos , Estrutura Secundária de Proteína , Rodopsina/química , Rodopsina/ultraestrutura , Transducina/química , Transducina/ultraestruturaRESUMO
The process of vision is initiated when the G protein-coupled receptor, rhodopsin (Rho), absorbs a photon and transitions to its activated Rho(∗) form. Rho(∗) binds the heterotrimeric G protein, transducin (G(t)) inducing GDP to GTP exchange and G(t) dissociation. Using nucleotide depletion and affinity chromatography, we trapped and purified the resulting nucleotide-free Rho(∗)·G(t) complex. Quantitative SDS-PAGE suggested a 2:1 molar ratio of Rho(∗) to G(t) in the complex and its mass determined by scanning transmission electron microscopy was 221±12kDa. A 21.6Å structure was calculated from projections of negatively stained Rho(∗)·G(t) complexes. The molecular envelope thus determined accommodated two Rho molecules together with one G(t) heterotrimer, corroborating the heteropentameric structure of the Rho(∗)·G(t) complex.
Assuntos
Complexos Multiproteicos/química , Rodopsina/química , Transducina/química , Eletroforese em Gel de Poliacrilamida , Guanosina Difosfato/química , Guanosina Trifosfato/química , Luz , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Multimerização Proteica , Rodopsina/ultraestrutura , Transducina/ultraestruturaRESUMO
The use of sub-nanometer resolution electron density as spatial constraints for de novo and ab initio structure prediction requires knowledge of protein boundaries to accurately segment the electron density for the prediction algorithms. Here we present a procedure where even poorly segmented density can be used to determine the fold of the protein. The method is automated, fast, capable of searching for multiple copies of a protein fold, and accessible to densities encompassing more than a thousand residues. The automation is particularly powerful as it allows the procedure to take full advantage of the expanding repository in the Protein Data Bank. We have tested the method on nine segmented sub-nanometer image reconstruction electron densities. The method successfully identifies the correct fold for the six densities for which an atomic structure is known, identifies a fold that agrees with prior structural data, a fold that agrees with predictions from the Fold & Function Assignment server, and a fold that correlates with secondary structure prediction. The identified folds in the last three examples can be used as templates for comparative modeling of the bacteriophage P22 tail-machine (a 3MDa complex composed of 39 protein subunits).