Production and Assay of Recombinant Multisubunit Chromatin Remodeling Complexes.

We have developed a novel system to facilitate the rapid and easy cloning of multiple genes (>10) in under a week. Using this system we have been able to successfully clone, overexpress, and purify a number of large multimeric proteins from insect cells, including the chromatin remodeling complexes SWR1 and INO80. Using Förster resonance energy transfer (FRET)-based assays we have demonstrated that our overexpressed enzymes have activities comparable to those purified from sources where the proteins are expressed under their endogenous promoters.

Functional characterization and architecture of recombinant yeast SWR1 histone exchange complex.

We have prepared recombinant fourteen subunit yeast SWR1 complex from insect cells using a modified MultiBac system. The 1.07 MDa recombinant protein complex has histone-exchange activity.

SIRT3 deacetylates ATP synthase F1 complex proteins in response to nutrient- and exercise-induced stress.

Aims: Adenosine triphosphate (ATP) synthase uses chemiosmotic energy across the inner mitochondrial membrane to convert adenosine diphosphate and orthophosphate into ATP, whereas genetic deletion of Sirt3 decreases mitochondrial ATP levels. Here, we investigate the mechanistic connection between SIRT3 and energy homeostasis.

Results: By using both in vitro and in vivo experiments, we demonstrate that ATP synthase F1 proteins alpha, beta, gamma, and Oligomycin sensitivity-conferring protein (OSCP) contain SIRT3-specific reversible acetyl-lysines that are evolutionarily conserved and bind to SIRT3.

Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F1-ATPase from bovine heart mitochondria.

The molecular description of the mechanism of F(1)-ATPase is based mainly on high-resolution structures of the enzyme from mitochondria, coupled with direct observations of rotation in bacterial enzymes. During hydrolysis of ATP, the rotor turns counterclockwise (as viewed from the membrane domain of the intact enzyme) in 120° steps. Because the rotor is asymmetric, at any moment the three catalytic sites are at different points in the catalytic cycle.

The structure of the membrane extrinsic region of bovine ATP synthase.

The structure of the complex between bovine mitochondrial F(1)-ATPase and a stator subcomplex has been determined at a resolution of 3.2 A. The resolved region of the stator contains residues 122-207 of subunit b; residues 5-25 and 35-57 of F(6); 3 segments of subunit d from residues 30-40, 65-74, and 85-91; and residues 1-146 and 169-189 of the oligomycin sensitivity conferral protein (OSCP).

Radiation exposure during elective coronary angioplasty: the effect of flat-panel detection.

Background: Coronary angiography and angioplasty have to date been performed using digital angiography and fluoroscopic systems which incorporate an image intensifier (II). More recently flat-panel (FP) detectors have been introduced which are thought to improve spatial resolution. However, there is limited data on the effect of flat-panel detection on radiation exposure.