Properties of lipids and the process of 2D crystallization of membrane proteins

International audienc

Phosphotyrosine-dependent in vitro reconstitution of recombinant LAT-nucleated multiprotein signalling complexes on liposomes.

Numerous cell surface receptors propagate activation signals to the interior of the cell via tyrosine phosphorylation of transmembrane proteins. This leads to the phosphotyrosine (PiY)-mediated recruitment of cytoplasmic signalling protein complexes which catalyze crucial biochemical signalling reactions. Here we describe the first in vitro reconstitution of such PiY-nucleated protein complexes on an artificial lipid membrane. A tyrosine phosphorylated recombinant variant of the transmembrane adaptor protein Linker for Activation of T cells (PiYLAT) was anchored in liposomes. These PiYLAT proteoliposomes specifically recruited cooperative high avidity signalling protein complexes from Jurkat cytosol. Nucleation of signalling protein assemblies readily occurred on PiYLAT liposomes composed of phosphatidylserine, but not on PiYLAT liposomes composed of phosphatidylcholine. Purified recombinant grb2 alone did not stably associate with tyrosine phosphorylated LAT proteoliposomes. However, when grb2 was presented to the PiYLAT proteoliposomes in the context of Jurkat cytosol it was incorporated into multiprotein signalling complexes. Together the data suggest that these reconstituted high-avidity signalling protein complexes represent a cooperative protein network. This novel in vitro approach offers a novel technology permitting biochemical, structural, and pharmacological analyses of plasma membrane receptor signalling complexes

Isolation, characterization and complete nucleotide sequence of a novel temperate bacteriophage Min1, isolated from the nematode pathogen Microbacterium nematophilum.

We report the discovery, properties and complete sequence (46,365bp) of Min1, the first bacteriophage to be reported for the coryneform genus Microbacterium. This temperate phage is normally integrated into a stable plasmid, pMN1, found in cells of Microbacterium nematophilum, a pathogen of certain soil nematodes including Caenorhabditis elegans, but it can also grow lytically. The phage is lambdoid in morphology and in sequence, belonging to the family Siphoviridae. General and specific features of the genome are discussed, together with possible contributions of the phage to host virulence

Synthesis of a hemifluorinated amphiphile designed for self-assembly and two-dimensional crystallization of membrane protein

The work reported herein deals with the synthesis and the preliminary physical-chemical analysis of new hemifluorinated surfactant made up of one fluorinated chain linked to a tricarboxylic acid polar head which is able to complex a Ni atom and should favor the two-dimensional crystallization of membrane proteins. Such a compound forms a Langmuir film which is a fluid at 20 °C and not perturbed by the presence of hydrocarbon detergent in aqueous solution. © 2008 Elsevier Ltd. All rights reserved

Calcineurin B-like domains in the large regulatory alpha/beta subunits of phosphorylase kinase.

Phosphorylase kinase (PhK) is a large hexadecameric complex that catalyzes the phosphorylation and activation of glycogen phosphorylase (GP). It consists in four copies each of a catalytic subunit (gamma) and three regulatory subunits (alpha beta delta). Delta corresponds to endogenous calmodulin, whereas little is known on the molecular architecture of the large alpha and beta subunits, which probably arose from gene duplication. Here, using sensitive methods of sequence analysis, we show that the C-terminal domain (named domain D) of these alpha and beta subunits can be significantly related to calcineurin B-like (CBL) proteins. CBL are members of the EF-hand family that are involved in the regulation of plant-specific kinases of the CIPK/PKS family, and relieve autoinhibition of their target kinases by binding to their regulatory region. The relationship highlighted here suggests that PhK alpha and/or beta domain D may be involved in a similar regulation mechanism, a hypothesis which is supported by the experimental observation of a direct interaction between domain D of PhKalpha and the regulatory region of the Gamma subunit. This finding, together the identification of significant similarities of domain D with the preceding domain C, may help to understand the molecular mechanism by which PhK alpha and/or beta domain D might regulate PhK activity