Jan Kvassman

High-resolution x-ray structure of human aquaporin 5

Human aquaporin 5 (HsAQP5) facilitates the transport of water across plasma membranes and has been identified within cells of the stomach, duodenum, pancreas, airways, lungs, salivary glands, sweat glands, eyes, lacrimal glands, and the inner ear. AQP5, like AQP2, is subject to posttranslational regulation by phosphorylation, at which point it is trafficked between intracellular storage compartments and the plasma membrane. Details concerning the molecular mechanism of membrane trafficking are unknown. Here we report the x-ray structure of HsAQP5 to 2.0-Å resolution and highlight structural similarities and differences relative to other eukaryotic aquaporins. A lipid occludes the putative central pore, preventing the passage of gas or ions through the center of the tetramer. Multiple consensus phosphorylation sites are observed in the structure and their potential regulatory role is discussed. We postulate that a change in the conformation of the C terminus may arise from the phosphorylation of AQP5 and thereby signal trafficking.

Stereochemistry of colchicinoids. Enantiomeric stability and binding to tubulin of desacetamidocolchicine and desacetamidoisocolchicine

Abstract Desacetamidocolchicine (3) and desacetamidoisocolchicine (4) have been chromatographically resolved into enantiomers. Thermal racemization gives inversion barriers of 22.1 and 23.4 kcal mol−1, respectively, for rotation around the bond joining the two aromatic rings. Kinetic binding experiments show that the enantiomer of 3 with the same helicity as native colchicine binds approximately 62 times as fast as colchicine to tubulin whereas the other enantiomer and both enantiomers of 4 do not bind. With molecular mechanics computations both the structures and the rotational barriers are reproduced, provided that the MM2(1985) force field, which gives stiffer aromatic rings than earlier force fields, is used.

Solute transport on the sub 100 ms scale across the lipid bilayer membrane of individual proteoliposomes

Screening assays designed to probe ligand and drug-candidate regulation of membrane proteins responsible for ion-translocation across the cell membrane are wide spread, while efficient means to screen membrane-protein facilitated transport of uncharged solutes are sparse. We report on a microfluidic-based system to monitor transport of uncharged solutes across the membrane of multiple (>100) individually resolved surface-immobilized liposomes. This was accomplished by rapidly switching (<10 ms) the solution above dye-containing liposomes immobilized on the floor of a microfluidic channel. With liposomes encapsulating the pH-sensitive dye carboxyfluorescein (CF), internal changes in pH induced by transport of a weak acid (acetic acid) could be measured at time scales down to 25 ms. The applicability of the set up to study biological transport reactions was demonstrated by examining the osmotic water permeability of human aquaporin (AQP5) reconstituted in proteoliposomes. In this case, the rate of osmotic-induced volume changes of individual proteoliposomes was time resolved by imaging the self quenching of encapsulated calcein in response to an osmotic gradient. Single-liposome analysis of both pure and AQP5-containing liposomes revealed a relatively large heterogeneity in osmotic permeability. Still, in the case of AQP5-containing liposomes, the single liposome data suggest that the membrane-protein incorporation efficiency depends on liposome size, with higher incorporation efficiency for larger liposomes. The benefit of low sample consumption and automated liquid handling is discussed in terms of pharmaceutical screening applications.