Your browser doesn't support javascript.
loading
Montrer: 20 | 50 | 100
Résultats 1 - 2 de 2
Filtrer
Plus de filtres











Base de données
Gamme d'année
1.
Cell Rep ; 11(8): 1184-92, 2015 May 26.
Article de Anglais | MEDLINE | ID: mdl-25981043

RÉSUMÉ

The membrane-spanning α helices of single-pass receptors play crucial roles in stabilizing oligomeric structures and transducing biochemical signals across the membrane. Probing intermolecular transmembrane interactions in single-pass receptors presents unique challenges, reflected in a gross underrepresentation of their membrane-embedded domains in structural databases. Here, we present two high-resolution structures of transmembrane assemblies from a eukaryotic single-pass protein crystallized in a lipidic membrane environment. Trimeric and tetrameric structures of the immunoreceptor signaling module DAP12, determined to 1.77-Å and 2.14-Å resolution, respectively, are organized by the same polar surfaces that govern intramembrane assembly with client receptors. We demonstrate that, in addition to the well-studied dimeric form, these trimeric and tetrameric structures are made in cells, and their formation is competitive with receptor association in the ER. The polar transmembrane sequences therefore act as primary determinants of oligomerization specificity through interplay between charge shielding and sequestration of polar surfaces within helix interfaces.


Sujet(s)
Protéines adaptatrices de la transduction du signal/composition chimique , Lipides membranaires/composition chimique , Protéines membranaires/composition chimique , Protéines adaptatrices de la transduction du signal/immunologie , Protéines adaptatrices de la transduction du signal/métabolisme , Séquence d'acides aminés , Cristallographie aux rayons X , Humains , Lipides membranaires/métabolisme , Protéines membranaires/immunologie , Protéines membranaires/métabolisme , Modèles moléculaires , Données de séquences moléculaires , Structure secondaire des protéines , Transduction du signal
2.
J Vis Exp ; (73): e50141, 2013 Mar 06.
Article de Anglais | MEDLINE | ID: mdl-23486227

RÉSUMÉ

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.


Sujet(s)
Disulfures/métabolisme , Protéines membranaires/biosynthèse , Protéines membranaires/composition chimique , Disulfures/composition chimique , Disulfures/isolement et purification , Escherichia coli/composition chimique , Escherichia coli/génétique , Escherichia coli/métabolisme , Humains , Protéines membranaires/génétique , Protéines membranaires/isolement et purification , Résonance magnétique nucléaire biomoléculaire/méthodes , Protéines recombinantes/biosynthèse , Protéines recombinantes/composition chimique , Protéines recombinantes/génétique , Protéines recombinantes/isolement et purification
SÉLECTION CITATIONS
DÉTAIL DE RECHERCHE