Structure and function of sodium-proton antiporters

Sodium-proton (Na+/H+) antiporters are secondary membrane protein transporters present in all living cells and are critical for sodium, pH and cell volume homeostasis. Their deregulation of transport activity has been linked to human diseases, such as, hypertension, heart failure and epilepsy and consequently they may be targets for drugs. In 2005, the first crystal structure of a Na+/H+ antiporter, NhaA from Escherichia coli, was solved at 3.45 Å resolution in an inward-facing conformation at pH 4 where the protein is inactive. Like many Na+/H+ antiporters, the activity of NhaA is regulated by pH and is active above pH 6.5. The main goal of this thesis was to solve a crystal structure of a Na+ /H+ antiporter in an active state. One of the problems in producing milligram quantities of purified membrane protein for crystallography is poor overexpression. For this reason, we first sought to improve membrane protein overexpression by developing a new expression platform, which we have called “MemStar” in E. coli using a test-case of control proteins, overall showing a boost in expression levels to at least 12 mg.L-1. This thesis describes the crystal structure of NapA from Thermus thermophilus, an NhaA homologue, which was solved to 3 Å in an outward-facing conformation at pH 7.8 in an active state. This NhaA homologue was selected as purified protein could grow better diffracting crystals than NhaA. The stability of NapA was also more suitable for purification in a small micelle detergent to improve diffracting resolution. Although NhaA crystals did not form above pH 6.5, a stabilised mutant was useful to confirm the position of a critical residue important in the mechanism. Structural comparisons with the NapA structure show the core domain moving relative to the dimerisation domain, similar to a rocking bundle model observed in other structures of different secondary active transporters sharing conserved structural features in their membrane protein folds also present in NhaA and NapA. This work has provided us with a fresh insight into the mechanism of Na+/H+ antiporters.Open Acces

End-grafted Sugar Chains as Aqueous Lubricant Additives: Synthesis and Macrotribological Tests of Poly( l -lysine)- graft -Dextran (PLL- g -dex) Copolymers

Comb-like graft copolymers with carbohydrate side chains have been developed as aqueous lubricant additives for oxide-based tribosystems, in an attempt to mimic biological lubrication systems, whose surfaces are known to be covered with sugar-rich layers. As adopted in the previous studies of the graft copolymer poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG), which showed both excellent lubricating and antifouling properties, a similar approach was chosen to graft dextran chains onto the same backbone, thus generating PLL-g-dex. PLL-g-dex copolymers readily adsorb from aqueous solution onto negatively charged oxide surfaces. Tribological characterization at the macroscopic scale, either under pure sliding conditions or a mixed sliding/rolling contact regime, shows that PLL-g-dex is very effective for the lubrication of oxide-based tribosystems. The relative lubricating capabilities of PLL-g-dex copolymers compared with PLL-g-PEG copolymers were observed to be highly dependent on the molecular structure of the copolymers (in particular, side-chain density along the backbone) and the measurement conditions (in particular, time between tribocontacts); the PLL-g-dex copolymers with a low degree of grafted side chains (≤20% grafting of available protonated primary amine groups along the backbone) showed better lubricating performance than their PLL-g-PEG counterparts at high tribocontact frequency (≥ca. 0.32Hz

Convex Hulls of Curves: Volumes and Signatures

Taking the convex hull of a curve is a natural construction in computational geometry. On the other hand, path signatures, central in stochastic analysis, capture geometric properties of curves, although their exact interpretation for levels larger than two is not well understood. In this paper, we study the use of path signatures to compute the volume of the convex hull of a curve. We present sufficient conditions for a curve so that the volume of its convex hull can be computed by such formulae. The canonical example is the classical moment curve, and our class of curves, which we call cyclic, includes other known classes such as $d$-order curves and curves with totally positive torsion. We also conjecture a necessary and sufficient condition on curves for the signature volume formula to hold. Finally, we give a concrete geometric interpretation of the volume formula in terms of lengths and signed areas.Comment: 15 pages, 5 figures. Comments are welcome

Inferring protein domain interactions from databases of interacting proteins

We describe domain pair exclusion analysis (DPEA), a method for inferring domain interactions from databases of interacting proteins. DPEA features a log odds score, E(ij), reflecting confidence that domains i and j interact. We analyzed 177,233 potential domain interactions underlying 26,032 protein interactions. In total, 3,005 high-confidence domain interactions were inferred, and were evaluated using known domain interactions in the Protein Data Bank. DPEA may prove useful in guiding experiment-based discovery of previously unrecognized domain interactions

Philanthropy patterns in major Australian performing arts organizations

Taking a management perspective in the field of philanthropy, this study examines 12 Australian major performing arts organizations over 19 years (2000–2018), which were identified as vulnerable and struggling with overreliance on public grants. Underpinned by theories that integrate understandings of external and internal resource management—resource dependence theory and the resource-based view—we uncover insights into what drives the increase in their philanthropic income. Using data from 228 annual reports and interviews, we present an original taxonomy that identifies organization-donor relationships and organizational efforts in nurturing philanthropy. We uncovered the interplays between donor engagement and positioning philanthropic staff in terms of organizational structure. Longitudinal financial and narrative data demonstrate that external resource management through donor engagement and internal resource management through organization structure emphasizing philanthropy have a significant impact on the growth of organizational philanthropic income