Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 62
Filtrar
1.
Cell ; 187(14): 3726-3740.e43, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38861993

RESUMO

Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.


Assuntos
Diferenciação Celular , Fatores de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos , Transdução de Sinais , Animais , Humanos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Camundongos , Ligantes , Cálcio/metabolismo , Sistema de Sinalização das MAP Quinases
2.
Annu Rev Cell Dev Biol ; 38: 125-153, 2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-35850151

RESUMO

The movement of lipids within and between membranes in bacteria is essential for building and maintaining the bacterial cell envelope. Moving lipids to their final destination is often energetically unfavorable and does not readily occur spontaneously. Bacteria have evolved several protein-mediated transport systems that bind specific lipid substrates and catalyze the transport of lipids across membranes and from one membrane to another. Specific protein flippases act in translocating lipids across the plasma membrane, overcoming the obstacle of moving relatively large and chemically diverse lipids between leaflets of the bilayer. Active transporters found in double-membraned bacteria have evolved sophisticated mechanisms to traffic lipids between the two membranes, including assembling to form large, multiprotein complexes that resemble bridges, shuttles, and tunnels, shielding lipids from the hydrophilic environment of the periplasm during transport. In this review, we explore our current understanding of the mechanisms thought to drive bacterial lipid transport.


Assuntos
Bactérias , Parede Celular , Transporte Biológico , Membrana Celular/metabolismo , Lipídeos/química
3.
Cell ; 181(3): 653-664.e19, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32359438

RESUMO

Gram-negative bacteria are surrounded by an outer membrane composed of phospholipids and lipopolysaccharide, which acts as a barrier and contributes to antibiotic resistance. The systems that mediate phospholipid trafficking across the periplasm, such as MCE (Mammalian Cell Entry) transporters, have not been well characterized. Our ~3.5 Å cryo-EM structure of the E. coli MCE protein LetB reveals an ~0.6 megadalton complex that consists of seven stacked rings, with a central hydrophobic tunnel sufficiently long to span the periplasm. Lipids bind inside the tunnel, suggesting that it functions as a pathway for lipid transport. Cryo-EM structures in the open and closed states reveal a dynamic tunnel lining, with implications for gating or substrate translocation. Our results support a model in which LetB establishes a physical link between the two membranes and creates a hydrophobic pathway for the translocation of lipids across the periplasm.


Assuntos
Proteínas de Bactérias/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/fisiologia , Transporte Biológico , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Transporte Proteico/fisiologia
4.
Cell ; 169(2): 273-285.e17, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28388411

RESUMO

How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Membrana/química , Membrana Celular/química , Cristalografia por Raios X , Microscopia Eletrônica , Modelos Moleculares , Complexos Multiproteicos/química
5.
Nature ; 627(8005): 898-904, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38480887

RESUMO

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures1. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints.


Assuntos
Nanoestruturas , Proteínas , Cristalografia por Raios X , Nanoestruturas/química , Proteínas/química , Proteínas/metabolismo , Microscopia Eletrônica , Reprodutibilidade dos Testes
6.
Cell ; 159(4): 857-68, 2014 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-25417161

RESUMO

Dyneins power microtubule motility using ring-shaped, AAA-containing motor domains. Here, we report X-ray and electron microscopy (EM) structures of yeast dynein bound to different ATP analogs, which collectively provide insight into the roles of dynein's two major ATPase sites, AAA1 and AAA3, in the conformational change mechanism. ATP binding to AAA1 triggers a cascade of conformational changes that propagate to all six AAA domains and cause a large movement of the "linker," dynein's mechanical element. In contrast to the role of AAA1 in driving motility, nucleotide transitions in AAA3 gate the transmission of conformational changes between AAA1 and the linker, suggesting that AAA3 acts as a regulatory switch. Further structural and mutational studies also uncover a role for the linker in regulating the catalytic cycle of AAA1. Together, these results reveal how dynein's two major ATP-binding sites initiate and modulate conformational changes in the motor domain during motility.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Dineínas/química , Dineínas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Catálise , Cristalografia por Raios X , Dictyostelium/química , Dineínas/ultraestrutura , Microscopia Eletrônica , Modelos Moleculares , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/ultraestrutura
7.
Nature ; 620(7973): 445-452, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37495693

RESUMO

To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host1,2. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis1,3, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids4-7 and cholesterol1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.


Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , Lipídeos , Proteínas de Membrana Transportadoras , Mycobacterium tuberculosis , Internalização do Vírus , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/ultraestrutura , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/ultraestrutura , Tuberculose/microbiologia , Fatores de Virulência/química , Fatores de Virulência/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Periplasma/metabolismo , Domínios Proteicos , Interações Hidrofóbicas e Hidrofílicas , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura
8.
Nature ; 616(7957): 581-589, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37020023

RESUMO

General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone1-3. Here, inspired by natural and re-engineered protein-peptide systems4-11, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone12. The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins.


Assuntos
Peptídeos , Engenharia de Proteínas , Proteínas , Sequência de Aminoácidos , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Engenharia de Proteínas/métodos , Ligação de Hidrogênio , Ligação Proteica , Dobramento de Proteína , Conformação Proteica
9.
Nat Chem Biol ; 20(7): 906-915, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38831036

RESUMO

Natural photosystems couple light harvesting to charge separation using a 'special pair' of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.


Assuntos
Clorofila , Clorofila/química , Clorofila/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Fotossíntese , Transferência de Energia , Microscopia Crioeletrônica , Conformação Proteica , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo
10.
Mol Ther ; 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956870

RESUMO

Several viruses hijack various forms of endocytosis in order to infect host cells. Here, we report the discovery of a molecule with antiviral properties that we named virapinib, which limits viral entry by macropinocytosis. The identification of virapinib derives from a chemical screen using high-throughput microscopy, where we identified chemical entities capable of preventing infection with a pseudotype virus expressing the spike (S) protein from SARS-CoV-2. Subsequent experiments confirmed the capacity of virapinib to inhibit infection by SARS-CoV-2, as well as by additional viruses, such as mpox virus and TBEV. Mechanistic analyses revealed that the compound inhibited macropinocytosis, limiting this entry route for the viruses. Importantly, virapinib has no significant toxicity to host cells. In summary, we present the discovery of a molecule that inhibits macropinocytosis, thereby limiting the infectivity of viruses that use this entry route such as SARS-CoV2.

11.
J Biol Chem ; 299(6): 104744, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37100290

RESUMO

The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer that protects the cell from external stressors, such as antibiotics. The Mla transport system is implicated in the Maintenance of OM Lipid Asymmetry by mediating retrograde phospholipid transport across the cell envelope. Mla uses a shuttle-like mechanism to move lipids between the MlaFEDB inner membrane complex and the MlaA-OmpF/C OM complex, via a periplasmic lipid-binding protein, MlaC. MlaC binds to MlaD and MlaA, but the underlying protein-protein interactions that facilitate lipid transfer are not well understood. Here, we take an unbiased deep mutational scanning approach to map the fitness landscape of MlaC from Escherichia coli, which provides insights into important functional sites. Combining this analysis with AlphaFold2 structure predictions and binding experiments, we map the MlaC-MlaA and MlaC-MlaD protein-protein interfaces. Our results suggest that the MlaD and MlaA binding surfaces on MlaC overlap to a large extent, leading to a model in which MlaC can only bind one of these proteins at a time. Low-resolution cryo-electron microscopy (cryo-EM) maps of MlaC bound to MlaFEDB suggest that at least two MlaC molecules can bind to MlaD at once, in a conformation consistent with AlphaFold2 predictions. These data lead us to a model for MlaC interaction with its binding partners and insights into lipid transfer steps that underlie phospholipid transport between the bacterial inner and OMs.


Assuntos
Proteínas de Escherichia coli , Metabolismo dos Lipídeos , Proteínas de Membrana Transportadoras , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Lipídeos de Membrana/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo
12.
J Biol Chem ; 299(12): 105321, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37802313

RESUMO

Staphylococcus aureus (S. aureus) is a serious global pathogen that causes a diverse range of invasive diseases. S. aureus utilizes a family of pore-forming toxins, known as bi-component leukocidins, to evade the host immune response and promote infection. Among these is LukAB (leukocidin A/leukocidin B), a toxin that assembles into an octameric ß-barrel pore in the target cell membrane, resulting in host cell death. The established cellular receptor for LukAB is CD11b of the Mac-1 complex. Here, we show that hydrogen voltage-gated channel 1 is also required for the cytotoxicity of all major LukAB variants. We demonstrate that while each receptor is sufficient to recruit LukAB to the plasma membrane, both receptors are required for maximal lytic activity. Why LukAB requires two receptors, and how each of these receptors contributes to pore-formation remains unknown. To begin to resolve this, we performed an alanine scanning mutagenesis screen to identify mutations that allow LukAB to maintain cytotoxicity without CD11b. We discovered 30 mutations primarily localized in the stem domains of LukA and LukB that enable LukAB to exhibit full cytotoxicity in the absence of CD11b. Using crosslinking, electron microscopy, and hydroxyl radical protein footprinting, we show these mutations increase the solvent accessibility of the stem domain, priming LukAB for oligomerization. Together, our data support a model in which CD11b binding unlatches the membrane penetrating stem domains of LukAB, and this change in flexibility promotes toxin oligomerization.


Assuntos
Proteínas de Bactérias , Leucocidinas , Staphylococcus aureus , Toxinas Biológicas , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Leucocidinas/genética , Leucocidinas/metabolismo , Leucocidinas/toxicidade , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Toxinas Biológicas/metabolismo , Mutação , Ligação Proteica/genética , Domínios Proteicos , Linhagem Celular , Células CHO , Cricetulus , Animais
13.
Proteins ; 91(12): 1571-1599, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37493353

RESUMO

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.


Assuntos
Biologia Computacional , Proteínas , Conformação Proteica , Modelos Moleculares , Biologia Computacional/métodos , Proteínas/química
14.
EMBO J ; 38(13): e101414, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31268607

RESUMO

The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer-binding, and mechanical elements. Such communication is particularly complex and not well understood in the dynein motor, an ATPase that is comprised of a ring of six AAA domains, a large mechanical element (linker) spanning over the ring, and a microtubule-binding domain (MTBD) that is separated from the AAA ring by a ~ 135 Å coiled-coil stalk. We identified mutations in the stalk that disrupt directional motion, have microtubule-independent hyperactive ATPase activity, and nucleotide-independent low affinity for microtubules. Cryo-electron microscopy structures of a mutant that uncouples ATPase activity from directional movement reveal that nucleotide-dependent conformational changes occur normally in one-half of the AAA ring, but are disrupted in the other half. The large-scale linker conformational change observed in the wild-type protein is also inhibited, revealing that this conformational change is not required for ATP hydrolysis. These results demonstrate an essential role of the stalk in regulating motor activity and coupling conformational changes across the two halves of the AAA ring.


Assuntos
Acetiltransferases/química , Acetiltransferases/metabolismo , Microtúbulos/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferases/genética , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
PLoS Pathog ; 16(9): e1008738, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946515

RESUMO

Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process of three human-infecting microsporidian species in vitro: Anncaliia algerae, Encephalitozoon hellem and E. intestinalis. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 µm⋅s-1, and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is at least 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection.


Assuntos
Microscopia/métodos , Microsporídios/patogenicidade , Organelas/imunologia , Organelas/ultraestrutura , Esporos Fúngicos/imunologia , Esporos Fúngicos/ultraestrutura , Proteínas Fúngicas/metabolismo , Microsporídios/imunologia , Microsporídios/ultraestrutura , Esporos Fúngicos/crescimento & desenvolvimento
16.
Proc Natl Acad Sci U S A ; 116(10): 4275-4284, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30770448

RESUMO

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil "stalk" of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Ionóforos/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Nanotecnologia/métodos , Cor , Dineínas/ultraestrutura , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Modelos Teóricos , Nanotecnologia/instrumentação , Sensibilidade e Especificidade , Fluxo de Trabalho
17.
Nature ; 528(7583): 580-4, 2015 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-26675729

RESUMO

A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.


Assuntos
Motivos de Aminoácidos , Bioengenharia , Simulação por Computador , Conformação Proteica , Proteínas/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dobramento de Proteína , Estabilidade Proteica , Temperatura
18.
Trends Biochem Sci ; 41(1): 94-105, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26678005

RESUMO

Cytoplasmic dynein, a member of the AAA (ATPases Associated with diverse cellular Activities) family of proteins, drives the processive movement of numerous intracellular cargos towards the minus end of microtubules. Here, we summarize the structural and motile properties of dynein and highlight features that distinguish this motor from kinesin-1 and myosin V, two well-studied transport motors. Integrating information from recent crystal and cryoelectron microscopy structures, as well as high-resolution single-molecule studies, we also discuss models for how dynein biases its movement in one direction along a microtubule track, and present a movie that illustrates these principles.


Assuntos
Dineínas/metabolismo , Microtúbulos/metabolismo , Animais , Dineínas/química , Humanos , Microtúbulos/química , Modelos Moleculares
19.
Nature ; 489(7417): 526-32, 2012 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-22982990

RESUMO

Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Vírus da Influenza A/classificação , Vírus da Influenza A/imunologia , Animais , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Especificidade de Anticorpos/genética , Antígenos Virais/química , Antígenos Virais/imunologia , Sítios de Ligação , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Sequência Conservada , Reações Cruzadas/genética , Reações Cruzadas/imunologia , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/química , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A/química , Vacinas contra Influenza/imunologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Conformação Proteica
20.
Proc Natl Acad Sci U S A ; 111(12): 4449-54, 2014 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-24591605

RESUMO

Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a protein's folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular protein's functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo.


Assuntos
Proteínas de Escherichia coli/metabolismo , Corantes Fluorescentes , Dobramento de Proteína , Trifosfato de Adenosina/metabolismo , Escherichia coli/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA