Hydrogenated Diglucose Detergents for Membrane-Protein Extraction and Stabilization.

We report herein the design and synthesis of a novel series of alkyl glycoside detergents consisting of a nonionic polar headgroup that comprises two glucose moieties in a branched arrangement (DG), onto which octane-, decane-, and dodecanethiols were grafted leading to ODG, DDG, and DDDG detergents, respectively. Micellization in aqueous solution was studied by isothermal titration calorimetry, 1H NMR spectroscopy, and surface tensiometry. Critical micellar concentration values were found to decrease by a factor of ∼10 for each pair of methylene groups added to the alkyl chain, ranging from ∼0.05 to 9 mM for DDDG and ODG, respectively. Dynamic light scattering and analytical ultracentrifugation sedimentation velocity experiments were used to investigate the size and composition of the micellar aggregates, showing that the aggregation number significantly increased from ∼40 for ODG to ∼80 for DDDG. All new compounds were able to solubilize membrane proteins (MPs) from bacterial membranes, insect cells, as well as the Madin-Darby canine kidney cells. In particular, native human adenosine receptor (A2AR) and bacterial transporter (BmrA) were solubilized efficiently. Striking thermostability improvements of +13 and +8 °C were observed when ODG and DDG were, respectively, applied to wild-type and full-length A2AR. Taken together, this novel detergent series shows promising detergent potency for solubilization and stabilization of membrane proteins (MPs) and thus makes a valuable addition to the chemical toolbox available for extracting and handling these important but challenging MP targets.

Molecular architecture of potassium chloride co-transporter KCC2.

KCC2 is a neuron specific K+-Cl- co-transporter that controls neuronal chloride homeostasis, and is critically involved in many neurological diseases including brain trauma, epilepsies, autism and schizophrenia. Despite significant accumulating data on the biology and electrophysiological properties of KCC2, structure-function relationships remain poorly understood. Here we used calixarene detergent to solubilize and purify wild-type non-aggregated and homogenous KCC2. Specific binding of inhibitor compound VU0463271 was demonstrated using surface plasmon resonance (SPR). Mass spectrometry revealed glycosylations and phosphorylations as expected from functional KCC2. We show by electron microscopy (EM) that KCC2 exists as monomers and dimers in solution. Monomers are organized into "head" and "core" domains connected by a flexible "linker". Dimers are asymmetrical and display a bent "S-shape" architecture made of four distinct domains and a flexible dimerization interface. Chemical crosslinking in reducing conditions shows that disulfide bridges are involved in KCC2 dimerization. Moreover, we show that adding a tag to the C-terminus is detrimental to KCC2 function. We postulate that the conserved KCC2 C-ter may be at the interface of dimerization. Taken together, our findings highlight the flexible multi-domain structure of KCC2 with variable anchoring points at the dimerization interface and an important C-ter extremity providing the first in-depth functional architecture of KCC2.

Thyroid function testing in an inpatient mental health unit.

OBJECTIVE: Thyroid function tests are a common screening investigation for patients admitted to a psychiatric inpatient unit. METHOD: This study aimed to retrospectively assess the clinical utility of routine thyroid function testing performed on newly admitted psychiatric patients over a 4-year period in Victoria, Australia via chart review of all abnormal results identified. RESULTS: Our retrospective audit revealed only two cases where identification of thyroid dysfunction informed patient management. In each case, the patient had a known history of thyroid disease. In this audit period, 893 patients required screening to yield one clinically relevant abnormal result, costing AU$24,975.57. CONCLUSION: Such low clinical utility does not support routine admission thyroid function tests for psychiatric inpatients. We conclude that thyroid function tests should only be performed where the history and clinical signs suggest a likely contribution of thyroid dysfunction to the psychiatric presentation.