Membrane Protein Production in Escherichia coli: Protocols and Rules.

Despite recent progresses in the use of eukaryotic expression system, production of membrane proteins for structural studies still relies on microbial expression systems. In this review, we provide protocols to achieve high level expression of membrane proteins in Escherichia coli, especially using the T7 RNA polymerase based expression system. From the design of the construct, the choice of the appropriate vector-host combination, the assessment of the bacterial fitness, to the selection of bacterial mutant adapted to the production of the target membrane protein, the chapter covers all necessary methods for a rapid optimization of a specific target membrane protein.

Exploration of the dynamic interplay between lipids and membrane proteins by hydrostatic pressure.

Cell membranes represent a complex and variable medium in time and space of lipids and proteins. Their physico-chemical properties are determined by lipid components which can in turn influence the biological function of membranes. Here, we used hydrostatic pressure to study the close dynamic relationships between lipids and membrane proteins.

Structural models of mitochondrial uncoupling proteins obtained in DPC micelles are not functionally relevant.

Uncoupling protein 1 (UCP1) is found in the inner mitochondrial membrane of brown adipocytes. In the presence of long-chain fatty acids (LCFAs), UCP1 increases the proton conductance, which, in turn, increases fatty acid oxidation and energy release as heat. Atomic models of UCP1 and UCP2 have been generated based on the NMR backbone structure of UCP2 in dodecylphosphocholine (DPC), a detergent known to inactivate UCP1.

Structure of the agonist 12-HHT in its BLT2 receptor-bound state.

G Protein-Coupled receptors represent the main communicating pathway for signals from the outside to the inside of most of eukaryotic cells. They define the largest family of integral membrane receptors at the surface of the cells and constitute the main target of the current drugs on the market. The low affinity leukotriene receptor BLT2 is a receptor involved in pro- and anti-inflammatory pathways and can be activated by various unsaturated fatty acid compounds.

NMR analysis of GPCR conformational landscapes and dynamics.

Understanding the signal transduction mechanism mediated by the G Protein-Coupled Receptors (GPCRs) in eukaryote cells represents one of the main issues in modern biology. At the molecular level, various biophysical approaches have provided important insights on the functional plasticity of these complex allosteric machines. In this context, X-ray crystal structures published during the last decade represent a major breakthrough in GPCR structural biology, delivering important information on the activation process of these receptors through the description of the three-dimensional organization of their active and inactive states.

Microbial expression systems for membrane proteins.

Despite many high-profile successes, recombinant membrane protein production remains a technical challenge; it is still the case that many fewer membrane protein structures have been published than those of soluble proteins. However, progress is being made because empirical methods have been developed to produce the required quantity and quality of these challenging targets. This review focuses on the microbial expression systems that are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies.

Functional Modulation of a G Protein-Coupled Receptor Conformational Landscape in a Lipid Bilayer.

Mapping the conformational landscape of G protein-coupled receptors (GPCRs), and in particular how this landscape is modulated by the membrane environment, is required to gain a clear picture of how signaling proceeds. To this end, we have developed an original strategy based on solution-state nuclear magnetic resonance combined with an efficient isotope labeling scheme. This strategy was applied to a typical GPCR, the leukotriene B4 receptor BLT2, reconstituted in a lipid bilayer.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting.

The Stable Interaction Between Signal Peptidase LepB of Escherichia coli and Nuclease Bacteriocins Promotes Toxin Entry into the Cytoplasm.

LepB is a key membrane component of the cellular secretion machinery, which releases secreted proteins into the periplasm by cleaving the inner membrane-bound leader. We showed that LepB is also an essential component of the machinery hijacked by the tRNase colicin D for its import. Here we demonstrate that this non-catalytic activity of LepB is to promote the association of the central domain of colicin D with the inner membrane before the FtsH-dependent proteolytic processing and translocation of the toxic tRNase domain into the cytoplasm.

Escherichia coli as host for membrane protein structure determination: a global analysis.

The structural biology of membrane proteins (MP) is hampered by the difficulty in producing and purifying them. A comprehensive analysis of protein databases revealed that 213 unique membrane protein structures have been obtained after production of the target protein in E. coli.

Structure of the prolyl-acyl carrier protein oxidase involved in the biosynthesis of the cyanotoxin anatoxin-a.

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins biosynthesized from L-proline by a short pathway involving polyketide synthases. Proline is first loaded onto AnaD, an acyl carrier protein, and prolyl-AnaD is then oxidized to 1-pyrroline-5-carboxyl-AnaD by a flavoprotein, AnaB. Three polyketide synthases then transform this imine into anatoxin-a or homoanatoxin-a.

Cyclopiazonic acid is complexed to a divalent metal ion when bound to the sarcoplasmic reticulum Ca2+-ATPase.

We have determined the structure of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in an E2.P(i)-like form stabilized as a complex with MgF(4)(2-), an ATP analog, adenosine 5'-(beta,gamma-methylene)triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.

Dimeric structure of human Na+/H+ exchanger isoform 1 overproduced in Saccharomyces cerevisiae.

The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H(+) in exchange for one extracellular Na(+). The human NHE1 isoform is involved in heart disease and cell growth and proliferation. Although details of NHE1 regulation and transport are being revealed, there is little information available on the structure of the intact protein.

The molecular basis for cyclopiazonic acid inhibition of the sarcoplasmic reticulum calcium pump.

The sarcoplasmic reticulum Ca(2+)-ATPase is essential for calcium reuptake in the muscle contraction-relaxation cycle. Here we present structures of a calcium-free state with bound cyclopiazonic acid (CPA) and magnesium fluoride at 2.65 A resolution and a calcium-free state with bound CPA and ADP at 3.

Regulation and functional roles of Grb14.

Grb14 is the last described member of the Grb7 family of adaptors, containing Grb7, Grb10 and Grb14. These proteins share a series of conserved domains involved in protein-protein and protein-lipid interactions: an amino terminal proline-rich region, a C-terminal SH2 domain, and a central GM region containing a RA, a PH domain, and a newly described PIR (BPS) region. As shown for the other members of the Grb7/10/14 family, Grb14 binds to various receptor tyrosine kinases (RTKs) under ligand induction.

The PIR domain of Grb14 is an intrinsically unstructured protein: implication in insulin signaling.

Grb14 belongs to the Grb7 family of adapter proteins and was identified as a negative regulator of insulin signal transduction. Its inhibitory effect on the insulin receptor kinase activity is controlled by a newly discovered domain called PIR. To investigate the biochemical and biophysical characteristics of this new domain, we cloned and purified recombinant PIR-SH2, PIR, and SH2 domains.