Structural analysis of a nanoparticle containing a lipid bilayer used for detergent-free extraction of membrane proteins.

In the past few years there has been a growth in the use of nano-particles for stabilizing lipid membranes with embedded proteins. These bionanoparticles provide a solution to the challenging problem of membrane protein isolation by maintaining a lipid bilayer essential to protein integrity and activity. We have described the use of an amphipathic polymer (Poly(styrene-co-maleic acid); SMA) to produce discoidal nanoparticles that contain a lipid bilayer with embedded protein.

Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain.

P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATP-Binding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds.

OSBPL10, a novel candidate gene for high triglyceride trait in dyslipidemic Finnish subjects, regulates cellular lipid metabolism.

Analysis of variants in three genes encoding oxysterol-binding protein (OSBP) homologues (OSBPL2, OSBPL9, OSBPL10) in Finnish families with familial low high-density lipoprotein (HDL) levels (N = 426) or familial combined hyperlipidemia (N = 684) revealed suggestive linkage of OSBPL10 single-nucleotide polymorphisms (SNPs) with extreme end high triglyceride (TG; >90th percentile) trait. Prompted by this initial finding, we carried out association analysis in a metabolic syndrome subcohort (Genmets) of Health2000 examination survey (N = 2,138), revealing association of multiple OSBPL10 SNPs with high serum TG levels (>95th percentile). To investigate whether OSBPL10 could be the gene underlying the observed linkage and association, we carried out functional experiments in the human hepatoma cell line Huh7.

siRNA screening reveals JNK2 as an evolutionary conserved regulator of triglyceride homeostasis.

Lipid homeostasis is essential for proper function of cells and organisms. To unravel new regulators of this system, we developed a screening procedure, combining RNA interference in HeLa cells and TLC, which enabled us to monitor modifications of lipid composition resulting from short, interfering RNA knock-downs. We applied this technique to the analysis of 600 human kinases.

Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra.

Direct profiling of total lipid extracts on a hybrid LTQ Orbitrap mass spectrometer by high-resolution survey spectra clusters species of 11 major lipid classes into 7 groups, which are distinguished by their sum compositions and could be identified by accurately determined masses. Rapid acquisition of survey spectra was employed as a "top-down" screening tool that, together with the computational method of principal component analysis, revealed pronounced perturbations in the abundance of lipid precursors within the entire series of experiments. Altered lipid precursors were subsequently identified either by accurately determined masses or by in-depth MS/MS characterization that was performed on the same instrument.

Phosphorylation-induced conformational changes of cystic fibrosis transmembrane conductance regulator monitored by attenuated total reflection-Fourier transform IR spectroscopy and fluorescence spectroscopy.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ABC protein superfamily. Phosphorylation of a regulatory domain of this protein is a prerequisite for activity. We analyzed the effect of protein kinase A (PKA) phosphorylation on the structure of purified and reconstituted CFTR protein.

A new experimental approach to detect long-range conformational changes transmitted between the membrane and cytosolic domains of LmrA, a bacterial multidrug transporter.

LmrA confers multidrug resistance to Lactococcus lactis by mediating the extrusion of antibiotics, out of the bacterial membrane, using the energy derived from ATP hydrolysis. Cooperation between the cytosolic and membrane-embedded domains plays a crucial role in regulating the transport ATPase cycle of this protein. In order to demonstrate the existence of a structural coupling required for the cross-talk between drug transport and ATP hydrolysis, we studied specifically the dynamic changes occurring in the membrane-embedded and cytosolic domains of LmrA by combining infrared linear dichroic spectrum measurements in the course of H/D exchange with Trp fluorescence quenching by a water-soluble attenuator.