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1.
Elife ; 122023 06 14.
Artículo en Inglés | MEDLINE | ID: mdl-37314846

RESUMEN

Guanylate binding proteins (GBPs) are soluble dynamin-like proteins that undergo a conformational transition for GTP-controlled oligomerization and disrupt membranes of intracellular parasites to exert their function as part of the innate immune system of mammalian cells. We apply neutron spin echo, X-ray scattering, fluorescence, and EPR spectroscopy as techniques for integrative dynamic structural biology to study the structural basis and mechanism of conformational transitions in the human GBP1 (hGBP1). We mapped hGBP1's essential dynamics from nanoseconds to milliseconds by motional spectra of sub-domains. We find a GTP-independent flexibility of the C-terminal effector domain in the µs-regime and resolve structures of two distinct conformers essential for an opening of hGBP1 like a pocket knife and for oligomerization. Our results on hGBP1's conformational heterogeneity and dynamics (intrinsic flexibility) deepen our molecular understanding relevant for its reversible oligomerization, GTP-triggered association of the GTPase-domains and assembly-dependent GTP-hydrolysis.


Asunto(s)
GTP Fosfohidrolasas , Proteínas de Unión al GTP , Animales , Humanos , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Hidrólisis , Guanosina Trifosfato/metabolismo , Biología , Mamíferos/metabolismo
2.
Curr Opin Struct Biol ; 40: 163-185, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27939973

RESUMEN

Förster Resonance Energy Transfer (FRET) combined with single-molecule spectroscopy probes macromolecular structure and dynamics and identifies coexisting conformational states. We review recent methodological developments in integrative structural modeling by satisfying spatial restraints on networks of FRET pairs (hybrid-FRET). We discuss procedures to incorporate prior structural knowledge and to obtain optimal distance networks. Finally, a workflow for hybrid-FRET is presented that automates integrative structural modeling and experiment planning to put hybrid-FRET on rails. To test this workflow, we simulate realistic single-molecule experiments and resolve three protein conformers, exchanging at 30µs and 10ms, with accuracies of 1-3Å RMSD versus the target structure. Incorporation of data from other spectroscopies and imaging is also discussed.


Asunto(s)
Bioquímica/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Modelos Moleculares , Animales , Humanos
3.
Sci Rep ; 6: 36792, 2016 11 11.
Artículo en Inglés | MEDLINE | ID: mdl-27833095

RESUMEN

TGR5 is the first identified bile acid-sensing G-protein coupled receptor, which has emerged as a potential therapeutic target for metabolic disorders. So far, structural and multimerization properties are largely unknown for TGR5. We used a combined strategy applying cellular biology, Multiparameter Image Fluorescence Spectroscopy (MFIS) for quantitative FRET analysis, and integrative modelling to obtain structural information about dimerization and higher-order oligomerization assemblies of TGR5 wildtype (wt) and Y111 variants fused to fluorescent proteins. Residue 111 is located in transmembrane helix 3 within the highly conserved ERY motif. Co-immunoprecipitation and MFIS-FRET measurements with gradually increasing acceptor to donor concentrations showed that TGR5 wt forms higher-order oligomers, a process disrupted in TGR5 Y111A variants. From the concentration dependence of the MFIS-FRET data we conclude that higher-order oligomers - likely with a tetramer organization - are formed from dimers, the smallest unit suggested for TGR5 Y111A variants. Higher-order oligomers likely have a linear arrangement with interaction sites involving transmembrane helix 1 and helix 8 as well as transmembrane helix 5. The latter interaction is suggested to be disrupted by the Y111A mutation. The proposed model of TGR5 oligomer assembly broadens our view of possible oligomer patterns and affinities of class A GPCRs.


Asunto(s)
Multimerización de Proteína , Receptores Acoplados a Proteínas G/metabolismo , Sustitución de Aminoácidos , Animales , Perros , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Modelos Moleculares , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de la Célula Individual , Espectrometría de Fluorescencia
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