RESUMO
Cilia are ubiquitous, hair-like appendages found in eukaryotic cells that carry out functions of cell motility and sensory reception. Cilia contain an intriguing cytoskeletal structure, termed the axoneme that consists of nine doublet microtubules radially interlinked and longitudinally organized in multiple specific repeat units. Little is known, however, about how the axoneme allows cilia to be both actively bendable and sturdy or how it is assembled. To answer these questions, we used cryo-electron microscopy to structurally analyse several of the repeating units of the doublet at sub-nanometre resolution. This structural detail enables us to unambiguously assign α- and ß-tubulins in the doublet microtubule lattice. Our study demonstrates the existence of an inner sheath composed of different kinds of microtubule inner proteins inside the doublet that likely stabilizes the structure and facilitates the specific building of the B-tubule.
Assuntos
Axonema/química , Cílios/química , Microtúbulos/química , Proteínas de Protozoários/química , Tetrahymena thermophila/química , Tubulina (Proteína)/química , Sequência de Aminoácidos , Axonema/ultraestrutura , Sítios de Ligação , Cílios/ultraestrutura , Microscopia Crioeletrônica , Citoesqueleto/química , Citoesqueleto/ultraestrutura , Flagelos/química , Flagelos/ultraestrutura , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tetrahymena thermophila/ultraestrutura , Termodinâmica , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
FAAP20 (Fanconi anemia-associated protein 20) is a subunit of the Fanconi anemia (FA) core complex that repairs interstrand cross-links. To understand the molecular basis for the FA core complex-mediated recruitment of Rev1 to the DNA lesion, we characterized the interactions among FAAP20-UBZ4, Rev1-BRCT, and ubiquitin using NMR. We found that FAAP20-UBZ4 binds not only ubiquitin but also Rev1-BRCT. Mapping the protein-protein interactions showed that FAAP20-UBZ4 has distinct binding surfaces for ubiquitin and Rev1-BRCT. In addition, the chemical exchange patterns indicated that the interaction between FAAP20-UBZ4 and ubiquitin might enhance the binding affinity between FAAP20-UBZ4 and Rev1-BRCT. These results provide new insight into the Rev1 recognition mechanism by FAAP20.
Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas Nucleares/química , Nucleotidiltransferases/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Sítios de Ligação/genética , Fenômenos Biofísicos , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Homologia de Sequência de Aminoácidos , Propriedades de Superfície , Ubiquitina/química , Ubiquitina/metabolismoRESUMO
Cnu is a nucleoid protein that has a high degree of sequence homology with Hha/YmoA family proteins, which bind to chromatin and regulate the expression of Escherichia coli virulence genes in response to changes in temperature or ionic strength. Here, we determined its solution structure and dynamic properties and mapped H-NS binding sites. Cnu consists of three alpha helices that are comparable with those of Hha, but it has significant flexibility in the C-terminal region and lacks a short alpha helix present in Hha. Upon increasing ionic strength, the helical structure of Cnu is destabilized, especially at the ends of the helices. The dominant H-NS binding sites, located at helix 3 as in Hha, reveal a common structural platform for H-NS binding. Our results may provide structural and dynamic bases for the similarity and dissimilarity between Cnu and Hha functions.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Dicroísmo Circular , Clonagem Molecular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Escherichia coli/química , Modelos Moleculares , Conformação Proteica , Dobramento de ProteínaRESUMO
Free-radical polymerization inside mesoporous silica has been investigated in order to open a route to functional polymer-silica composite materials with well-defined mesoporosity. Various vinyl monomers, such as styrene, chloromethyl styrene, 2-hydroxyethyl methacrylate, and methacrylic acid, were polymerized after impregnation into mesoporous silicas with various structures, which were synthesized using polyalkylene oxide-type block copolymers. The location of the polymers was systematically controlled with detailed structures of the silica framework and the polymerization conditions. Particularly noteworthy is the polymer-silica composite structure obtained by in situ polymerization after the selective adsorption of monomers as a uniform film on silica walls. The analysis of XRD data and the N(2) adsorption isotherms indicates the formation of uniform polymer nanocoating. The resultant polymer-silica composite materials can easily be post-functionalized to incorporate diverse functional groups in high density, due to the open porous structure allowing facile access for the chemical reagent. The fundamental characteristics of the composite materials are substantiated by testing the biomolecule's adsorption capacity and catalytic reactivity. Depending on the structure and composition of polymers, the resultant polymer-silica composite materials exhibit notably distinct adsorption properties toward biomolecules, such as proteins. Furthermore, it is demonstrated that the nanocoatings of polymers deposited on the mesopore walls have remarkably enhanced catalytic activity and selectivity, as compared to that of bulk polymer resins. We believe that, due to facile functionalization and attractive textural properties, the mesoporous polymer-silica composite materials are very useful for applications, such as adsorption, separation, host-guest complexes, and catalysis.