A rapid expression and purification condition screening protocol for membrane protein structural biology

Membrane proteins control a large number of vital biological processes and are often medically important—not least as drug targets. However, membrane proteins are generally more difficult to work with than their globular counterparts, and as a consequence comparatively few high‐resolution structures are available. In any membrane protein structure project, a lot of effort is usually spent on obtaining a pure and stable protein preparation. The process commonly involves the expression of several constructs and homologs, followed by extraction in various detergents. This is normally a time‐consuming and highly iterative process since only one or a few conditions can be tested at a time. In this article, we describe a rapid screening protocol in a 96‐well format that largely mimics standard membrane protein purification procedures, but eliminates the ultracentrifugation and membrane preparation steps. Moreover, we show that the results are robustly translatable to large‐scale production of detergent‐solubilized protein for structural studies. We have applied this protocol to 60 proteins from an E. coli membrane protein library, in order to find the optimal expression, solubilization and purification conditions for each protein. With guidance from the obtained screening data, we have also performed successful large‐scale purifications of several of the proteins. The protocol provides a rapid, low cost solution to one of the major bottlenecks in structural biology, making membrane protein structures attainable even for the small laboratory.     

Genetic variability affects the response of skeletal muscle to disuse

Objective:To examine whether genetic variability plays a role in skeletal muscle response to disuse.Methods:We examined skeletal muscle response to disuse in five different strains of mice: CAST/EiJ, NOD/ShiLtJ, NZO/HILtJ, 129S1/SvImJ and A/J. Mice had one limb immobilized by a cast for three weeks.Results:Response to immobilization was dependent on the strain of mice. Skeletal muscle mass/body weight was decreased by immobilization in all strains except 1291/SvImJ. Immobilization decreased absolute skeletal muscle mass in quadriceps and gastrocnemius in NOD/ShiltJ and NZO/HILtJ mice. Three weeks of immobilization resulted in an increase in quadriceps levels of atrogenes in CAST/EiJ. Immobilization resulted in an increase in quadriceps and gastrocnemius levels of Myh4 in CAST/EiJ. A similar trend was observed for Myh7 in gastrocnemius muscle. Immobilization resulted in a decrease of the p-p70S6K1/total p706SK1 ratio in quadriceps of NOD/ShiLtJ mice and the gastrocnemius of A/J mice. Immobilization did not affect the p-4EBP1/total 4EBP1 ratio in quadriceps of any of the strains examined. However, the p-4EBP1/total 4EBP1 ratio in gastrocnemius was greater in immobilized, relative to control, limbs in CAST/EiJ mice.Conclusion:Genetic variability affects the response of skeletal muscle to disuse.     

Heterogeneity in cancer cells: variation in drug response in different primary and secondary colorectal cancer cell lines in vitro

Tumor heterogeneity may give rise to differential responses to chemotherapy drugs. Therefore, unraveling tumor heterogeneity has an implication for biomarker discovery and cancer therapeutics. To test this phenomenon, we investigated the differential responses of three secondary colorectal cancer cell lines of different origins (HCT116, HT29, and SW620 cells) and four novel primary cell lines obtained from different colorectal cancer patients to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP) and explored the differences in gene expression among the primary cell lines in response to exposure to cytotoxic drugs. Cells were exposed to different doses of 5-FU and L-OHP separately or in combinations of equitoxic drug or equimolar drug ratios (median effect of Chou-Talalay principle). Cell viability was assessed using MTT assay and the respective IC₅₀ values were determined. Changes in gene expression in primary cell lines after exposure to the same drug doses were compared using real-time PCR array. The sensitivities (IC₅₀) of different cell lines, both secondary and primary, to 5-FU and L-OHP were significantly different, whether in monotherapy or combined treatment. Primary cell lines needed higher doses to reach IC₅₀. There were variations in gene expression among the primary cell lines of different chemosensitivities to the challenge of the same combined dose of 5-FU and L-OHP. The results confirm the heterogeneous nature of colorectal cancer cells from different patient tumors. Studies using primary cancer cells established from patient’s tumors rather than secondary cell lines will more closely reflect the actual character of the disease.     

Don't forget to look down – collaborative approaches to predator conservation

Finding effective ways of conserving large carnivores is widely recognised as a priority in conservation. However, there is disagreement about the most effective way to do this, with some favouring top‐down ‘command and control’ approaches and others favouring collaboration. Arguments for coercive top‐down approaches have been presented elsewhere; here we present arguments for collaboration. In many parts of the developed world, flexibility of approach is built into the legislation, so that conservation objectives are balanced with other legitimate goals. In the developing world, limited resources, poverty and weak governance mean that collaborative approaches are likely to play a particularly important part in carnivore conservation. In general, coercive policies may lead to the deterioration of political legitimacy and potentially to non‐compliance issues such as illegal killing, whereas collaborative approaches may lead to psychological ownership, enhanced trust, learning, and better social outcomes. Sustainable hunting/trapping plays a crucial part in the conservation and management of many large carnivores. There are many different models for how to conserve carnivores effectively across the world, research is now required to reduce uncertainty and examine the effectiveness of these approaches in different contexts.     

Formation of transition metal-doxorubicin complexes inside liposomes

Doxorubicin complexation with the transition metal manganese (Mn²⁺) has been characterized, differentiating between the formation of a doxorubicin-metal complex and doxorubicin fibrous-bundle aggregates typically generated following ion gradient-based loading procedures that rely on liposome encapsulated citrate or sulfate salts. The physical and chemical characteristics of the encapsulated drug were assessed using cryo-electron microscopy, circular dichroism (CD) and absorbance spectrophotometric analysis. In addition, in vitro and in vivo drug loading and release characteristics of the liposomal formulations were investigated. Finally, the internal pH after drug loading was measured with the aim of linking formation of the Mn²⁺ complex to the presence or absence of a transmembrane pH gradient. Doxorubicin was encapsulated into either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol (Chol) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/Chol liposomes, where the entrapped salts were citrate, MnSO₄ or MnCl₂. In response to a pH gradient or a Mn²⁺ ion gradient, doxorubicin accumulated inside to achieve a drug-to-lipid ratio of approximately 0.2:1 (wt/wt). Absorbance and CD spectra of doxorubicin in the presence of Mn²⁺ suggested that there are two distinct structures captured within the liposomes. In the absence of added ionophore A23187, drug loading is initiated on the basis of an established pH gradient; however, efficient drug uptake is not dependent on maintenance of the pH gradient. Drug release from DMPC/Chol is comparable regardless of whether doxorubicin is entrapped as a citrate-based aggregate or a Mn²⁺ complex. However, in vivo drug release from DSPC/Chol liposomes indicate less than 5% or greater than 50% drug loss over a 24-h time course when the drug was encapsulated as an aggregate or a Mn²⁺ complex, respectively. These studies define a method for entrapping drugs possessing coordination sites capable of complexing transition metals and suggest that drug release is dependent on lipid composition, internal pH, as well as the nature of the crystalline precipitate, which forms following encapsulation.     

Isolation and characterization of a thioredoxin-dependent peroxidase from Chlamydomonas reinhardtii.

All living organisms contain redox systems involving thioredoxins (Trx), proteins featuring an extremely conserved and reactive active site that perform thiol-disulfide interchanges with disulfide bridges of target proteins. In photosynthetic organisms, numerous isoforms of Trx coexist, as revealed by sequencing of Arabidopsis genome. The specific functions of many of them are still unknown. In an attempt to find new molecular targets of Trx in Chlamydomonas reinhardtii, an affinity column carrying a cytosolic Trx h mutated at the less reactive cysteine of its active site was used to trap Chlamydomonas proteins that form mixed disulfides with Trx. The major protein bound to the column was identified by amino-acid sequencing and mass spectrometry as a thioredoxin-dependent 2Cys peroxidase. Isolation and sequencing of its gene revealed that this peroxidase is most likely a chloroplast protein with a high homology to plant 2Cys peroxiredoxins. It is shown that the Chlamydomonas peroxiredoxin (Ch-Prx1) is active with various thioredoxin isoforms, functions as an antioxidant toward reactive oxygen species (ROS), and protects DNA against ROS-induced degradation. Expression of the peroxidase gene in Chlamydomonas was found to be regulated by light, oxygen concentration, and redox state. The data suggest a role for the Chlamydomonas Prx in ROS detoxification in the chloroplast.     

Interaction of decorin with CNBr peptides from collagens I and II. Evidence for multiple binding sites and essential lysyl residues in collagen.

Decorin is a small leucine-rich chondroitin/dermatan sulfate proteoglycan reported to interact with fibrillar collagens through its protein core and to localize at d and e bands of the collagen fibril banding pattern. Using a solid-phase assay, we have determined the interaction of peptides derived by CNBr cleavage of type I and type II collagen with decorin extracted from bovine tendon and its protein core and with a recombinant decorin preparation. At least five peptides have been found to interact with all three decorin samples. The interaction of peptides with tendon decorin has a dissociation constant in the nanomolar range. The triple helical conformation of the peptide trimeric species is a necessary requisite for the binding. All positive peptides have a region within the d and e bands of collagen fibrils. Two chemical derivatives of collagens and of positive peptides were prepared by N-acetylation and N-methylation of the primary amino group of Lys/Hyl side chains. Chemical modifications performed in mild conditions do not significantly alter the thermal stability of peptide trimeric species whereas they affect the interaction with decorin: N-acetylation eliminates both the positive charge and the binding to decorin, whereas N-methylation preserves the cationic character and modulates the binding. We conclude that decorin makes contacts with multiple sites in type I collagen and probably also in type II collagen and that some collagen Lys/Hyl residues are essential for the binding.     

Mate choice in a hermaphrodite: you won't score with a spermatophore

Sexual selection has played a major role in shaping the wide variety of mating patterns found in species with separate sexes, but little is known about its effects on simultaneous hermaphrodites. However, many hermaphrodites possess complex reproductive systems and mating behaviour is often elaborate, suggesting that some form of mate assessment takes place. We found that the marine slug Aeolidiella glauca, a simultaneous hermaphrodite with reciprocal external sperm transfer via spermatophores, shows a unique mate choice behaviour by avoiding mating with conspecifics already carrying a spermatophore received during the previous mating. Current mating status did not seem to affect this behaviour, because both slugs that had mated 2-3 days before our mate choice trials and slugs that had been isolated for 4-6 weeks avoided spermatophore-carrying partners. There are two obvious reasons why slugs should avoid recently mated partners. First, they may reduce the risk of getting a partner depleted in self-sperm. Second, the risk of sperm competition may be decreased. Histological investigations of sperm reserves suggest that sperm depletion did not influence our mate choice experiments. Most slugs had sufficient sperm stored for spermatophore production. Therefore, the most likely explanation for A. glauca's peculiar mate choice is that, by avoiding a recently mated partner, a sperm donor may reduce its risk of being subjected to sperm competition.     

Surface interactions in the complex between cytochrome f and the E43Q/D44N and E59K/E60Q plastocyanin double mutants as determined by 1H-NMR chemical shift analysis

A combination of site-directed mutagenesis and NMR chemical shift perturbation analysis of backbone and side-chain protons has been used to characterize the transient complex of the photosynthetic redox proteins plastocyanin and cytochrome f. To elucidate the importance of charged residues on complex formation, the complex of cytochrome f and E43Q/D44N or E59K/E60Q spinach plastocyanin double mutants was studied by full analysis of the (1)H chemical shifts by use of two-dimensional homonuclear NMR spectra. Both mutants show a significant overall decrease in chemical shift perturbations compared with wild-type plastocyanin, in agreement with a large decrease in binding affinity. Qualitatively, the E43Q/D44N mutant showed a similar interaction surface as wild-type plastocyanin. The interaction surface in the E59K/E60Q mutant was distinctly different from wild type. It is concluded that all four charged residues contribute to the affinity and that residues E59 and E60 have an additional role in fine tuning the orientation of the proteins in the complex.