Exploring the K+ binding site and its coupling to transport in the neurotransmitter:sodium symporter LeuT.

The neurotransmitter:sodium symporters (NSSs) are secondary active transporters that couple the reuptake of substrate to the symport of one or two sodium ions. One bound Na+ (Na1) contributes to the substrate binding, while the other Na+ (Na2) is thought to be involved in the conformational transition of the NSS. Two NSS members, the serotonin transporter (SERT) and the Drosophila dopamine transporter (dDAT), also couple substrate uptake to the antiport of K+ by a largely undefined mechanism. We have previously shown that the bacterial NSS homologue, LeuT, also binds K+, and could therefore serve as a model protein for the exploration of K+ binding in NSS proteins. Here, we characterize the impact of K+ on substrate affinity and transport as well as on LeuT conformational equilibrium states. Both radioligand binding assays and transition metal ion FRET (tmFRET) yielded similar K+ affinities for LeuT. K+ binding was specific and saturable. LeuT reconstituted into proteoliposomes showed that intra-vesicular K+ dose-dependently increased the transport velocity of [3H]alanine, whereas extra-vesicular K+ had no apparent effect. K+ binding induced a LeuT conformation distinct from the Na+- and substrate-bound conformation. Conservative mutations of the Na1 site residues affected the binding of Na+ and K+ to different degrees. The Na1 site mutation N27Q caused a >10-fold decrease in K+ affinity but at the same time a ~3-fold increase in Na+ affinity. Together, the results suggest that K+ binding to LeuT modulates substrate transport and that the K+ affinity and selectivity for LeuT is sensitive to mutations in the Na1 site, pointing toward the Na1 site as a candidate site for facilitating the interaction with K+ in some NSSs.

Rational Approach to Improve Detergent Efficacy for Membrane Protein Stabilization.

Membrane protein structures are essential for the molecular understanding of diverse cellular processes and drug discovery. Detergents are not only widely used to extract membrane proteins from membranes but also utilized to preserve native protein structures in aqueous solution. However, micelles formed by conventional detergents are suboptimal for membrane protein stabilization, necessitating the development of novel amphiphilic molecules with enhanced protein stabilization efficacy. In this study, we prepared two sets of tandem malonate-derived glucoside (TMG) variants, both of which were designed to increase the alkyl chain density in micelle interiors. The alkyl chain density was modulated either by reducing the spacer length (TMG-Ms) or by introducing an additional alkyl chain between the two alkyl chains of the original TMGs (TMG-Ps). When evaluated with a few membrane proteins including a G protein-coupled receptor, TMG-P10,8 was found to be substantially more efficient at extracting membrane proteins and also effective at preserving protein integrity in the long term compared to the previously described TMG-A13. This result reveals that inserting an additional alkyl chain between the two existing alkyl chains is an effective way to optimize detergent properties for membrane protein study. This new biochemical tool and the design principle described have the potential to facilitate membrane protein structure determination.

Ethnic discrimination in Scandinavia: evidence from a field experiment in women's amateur soccer.

In this paper, we examine ethnic discrimination using sport as a laboratory. Applying a field experiment in the three Scandinavian countries-Sweden, Norway, and Denmark-we test whether foreign female minority groups experience greater rejection rates when seeking inclusion in amateur soccer clubs. Soccer coaches were contacted by e-mail using native and foreign-sounding names from selected groups, requesting to participate in trial practice. Previous findings show persistent discrimination of foreign minority groups in the labour market, and recent work suggests that discrimination also occurs in the context of soccer. Our findings from Scandinavia show that Sweden is the only country that shows statistically significant signs of discriminatory patterns, and the probability of experiencing discrimination increases with cultural distance. However, cultural distance appears to have no influence in Norway and Denmark. We further investigate whether male or female coaches demonstrate different discriminatory behaviour when being contacted, but our analysis shows almost no gender differences. Findings suggest that how men and women differ in their discriminatory behaviour is context specific. The differences identified across nations and previous studies are discussed to better understand the mechanisms of discrimination.

Maltose-bis(hydroxymethyl)phenol (MBPs) and Maltose-tris(hydroxymethyl)phenol (MTPs) Amphiphiles for Membrane Protein Stability.

Membrane protein structures provide a fundamental understanding of their molecular actions and are of importance for drug development. Detergents are widely used to solubilize, stabilize, and crystallize membrane proteins, but membrane proteins solubilized in conventional detergents are prone to denaturation and aggregation. Thus, developing novel detergents with enhanced efficacy for protein stabilization remains important. We report herein the design and synthesis of a class of phenol-derived maltoside detergents. Using two different linkers, we prepared two sets of new detergents, designated maltose-bis(hydroxymethyl)phenol (MBPs) and maltose-tris(hydroxymethyl)phenol (MTPs). The evaluation of these detergents with three transporters and two G-protein coupled receptors allowed us to identify a couple of new detergents (MBP-C9 and MTP-C12) that consistently conferred enhanced stability to all tested proteins compared to a gold standard detergent (DDM). Furthermore, the data analysis based on the detergent structures provides key detergent features responsible for membrane protein stabilization that together will facilitate the future design of novel detergents.

Conformationally flexible core-bearing detergents with a hydrophobic or hydrophilic pendant: Effect of pendant polarity on detergent conformation and membrane protein stability.

Membrane protein structures provide atomic level insight into essential biochemical processes and facilitate protein structure-based drug design. However, the inherent instability of these bio-macromolecules outside lipid bilayers hampers their structural and functional study. Detergent micelles can be used to solubilize and stabilize these membrane-inserted proteins in aqueous solution, thereby enabling their downstream characterizations. Membrane proteins encapsulated in detergent micelles tend to denature and aggregate over time, highlighting the need for development of new amphiphiles effective for protein solubility and stability. In this work, we present newly-designed maltoside detergents containing a pendant chain attached to a glycerol-decorated tris(hydroxymethyl)methane (THM) core, designated GTMs. One set of the GTMs has a hydrophobic pendant (ethyl chain; E-GTMs), and the other set has a hydrophilic pendant (methoxyethoxylmethyl chain; M-GTMs) placed in the hydrophobic-hydrophilic interfaces. The two sets of GTMs displayed profoundly different behaviors in terms of detergent self-assembly and protein stabilization efficacy. These behaviors mainly arise from the polarity difference between two pendants (ethyl and methoxyethoxylmethyl chains) that results in a large variation in detergent conformation between these sets of GTMs in aqueous media. The resulting high hydrophobic density in the detergent micelle interior is likely responsible for enhanced efficacy of the M-GTMs for protein stabilization compared to the E-GTMs and a gold standard detergent DDM. A representative GTM, M-GTM-O12, was more effective for protein stability than some recently developed detergents including LMNG. This is the first case study investigating the effect of pendant polarity on detergent geometry correlated with detergent efficacy for protein stabilization. STATEMENT OF SIGNIFICANCE: This study introduces new amphiphiles for use as biochemical tools in membrane protein studies. We identified a few hydrophilic pendant-bearing amphiphiles such as M-GTM-O11 and M-GTM-O12 that show remarkable efficacy for membrane protein solubilization and stabilization compared to a gold standard DDM, the hydrophobic counterparts (E-GTMs) and a significantly optimized detergent LMNG. In addition, detergent results obtained in the current study reveals the effect of detergent pendant polarity on protein solubility and stability. Thus, the current study represents both significant chemical and conceptual advance. The detergent tools and design principle introduced here advance protein science and facilitate structure-based drug design and development.

New Malonate-Derived Tetraglucoside Detergents for Membrane Protein Stability.

Membrane proteins are widely studied in detergent micelles, a membrane-mimetic system formed by amphiphilic compounds. However, classical detergents have serious limitations in their utility, particularly for unstable proteins such as eukaryotic membrane proteins and membrane protein complexes, and thus, there is an unmet need for novel amphiphiles with enhanced ability to stabilize membrane proteins. Here, we developed a new class of malonate-derived detergents with four glucosides, designated malonate-derived tetra-glucosides (MTGs), and compared these new detergents with previously reported octyl glucose neopentyl glycol (OGNG) and n-dodecyl-ß-d-maltoside (DDM). When tested with two G-protein coupled receptors (GPCRs) and three transporters, a couple of MTGs consistently conferred enhanced stability to all tested proteins compared to DDM and OGNG. As a result of favorable behaviors for a range of membrane proteins, these MTGs have substantial potential for membrane protein research. This study additionally provides a new detergent design principle based on the effect of a polar functional group (i.e., ether) on protein stability depending on its position in the detergent scaffold.

Cycling-related injuries at Sørlandet Hospital, Kristiansand. / Sykkelrelaterte skader på Sørlandet sykehus Kristiansand.

BACKGROUND: It is a policy objective to increase the percentage of journeys made by bicycle in Norway from the current 5 % to 10 %. Kristiansand is one of the most active cities in Norway in terms of cycling. We wished to identify the extent of injuries among cyclists admitted to the hospital. MATERIAL AND METHOD: We reviewed the medical records of patients with cycling-related injuries who were admitted to Sørlandet Hospital, Kristiansand in the period 1 January 2012 to 31 December 2015. Patient, accident, injury and treatment characteristics were recorded, as well as any sequelae after 12 months. RESULTS: Altogether 224 adults and 53 children (<16 years) were registered with cycling-related injuries, most of which (n=192, 69 %) were mild/moderate. Very severe and critical injuries were recorded in 6 (11 %) children and 22 (10 %) adults. Fractures (n=179, 65 %) and minor head injuries (n= 78, 28 %) dominated the injury panorama. Surgical treatment was undertaken in 107 (48 %) adults and 19 (36 %) children. A total of 12 (4 %) patients were transferred to the trauma centre at Oslo University Hospital Ullevål. Four adults had significant sequelae after 12 months, all related to severe head/neck injury. INTERPRETATION: A considerable proportion of serious and complex injuries require that the national guidelines for use of a trauma team be followed. Systematic and ongoing registration of cyclists' injuries in the form of a national registry could help increase our insight into the circumstances surrounding accidents and the extent of injuries related to these.