A lower X-gate in TASK channels traps inhibitors within the vestibule.

TWIK-related acid-sensitive potassium (TASK) channels-members of the two pore domain potassium (K2P) channel family-are found in neurons1, cardiomyocytes2-4 and vascular smooth muscle cells5, where they are involved in the regulation of heart rate6, pulmonary artery tone5,7, sleep/wake cycles8 and responses to volatile anaesthetics8-11. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli12-15. Unlike other K2P channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation16. In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K2P channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate-which we designate as an 'X-gate'-created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues (243VLRFMT248) that are essential for responses to volatile anaesthetics10, neurotransmitters13 and G-protein-coupled receptors13. Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders.

The adaptability of the ion-binding site by the Ag(I)/Cu(I) periplasmic chaperone SilF.

The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram-negative bacteria. Sil proteins also bind Cu(I) but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry, we show that binding of both ions is not only tighter than previously thought but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram-negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment.

A review of the approaches used to solve sub-100 kDa membrane proteins by cryo-electron microscopy.

Membrane proteins (MPs) are essential components of all biological membranes, contributing to key cellular functions that include signalling, molecular transport and energy metabolism. Consequently, MPs are important biomedical targets for therapeutics discovery. Despite hardware and software developments in cryo-electron microscopy, as well as MP sample preparation, MPs smaller than 100 kDa remain difficult to study structurally. Significant investment is required to overcome low levels of naturally abundant protein, MP hydrophobicity as well as conformational and compositional instability. Here we have reviewed the sample preparation approaches that have been taken to successfully express, purify and prepare small MPs for analysis by cryo-EM (those with a total solved molecular weight of under 100 kDa), as well as examining the differing approaches towards data processing and ultimately obtaining a structural solution. We highlight common challenges at each stage in the process as well as strategies that have been developed to overcome these issues. Finally, we discuss future directions and opportunities for the study of sub-100 kDa membrane proteins by cryo-EM.

The role of protein hydrolysates in prolonging viability and enhancing antibody production of CHO cells.

Four independent mAb-producing CHO cell lines were grown in media supplemented with one of seven protein hydrolysates of animal and plant origin. This generated a 7x4 matrix of replicate cultures which was analysed for viable cell density and mAb productivity. In all cultures, a consistent growth rate was shown in batch culture up to 4 to 5 days. Differences between cultures appeared in the decline phase which was followed up to 7 days beyond the start of the cultures. There was a marginal but significant overall increase (x1.1) in the integral viable cell density (IVCD) in the presence of hydrolysate but a more substantial increase in the cell-specific mAb (qMab) productivity (x1.5). There were individual differences between hydrolysates in terms of enhancement of mAb productivity, the highest being a 166% increase of mAb titre (to 117 mg/L) in batch cultures of CHO-EG2 supplemented with UPcotton hydrolysate. The effect of one of the most active hydrolysates (HP7504) on antibody glycosylation was investigated. This showed no change in the predominant seven glycans produced but a significant increase in the galactosylation and sialylation of some but not all the antibodies. Overall, the animal hydrolysate, Primatone and two cotton-derived hydrolysates provided the most substantial benefit for enhanced productivity. The cotton-based hydrolysates can be viewed as valuable supplements for animal-derived component-free (ADCF) media and as a source for the investigation of chemically defined bioactive components. KEY POINTS: • Protein hydrolysates enhanced both IVCD & qMab; the effect on qMab being consistently greater. • Cotton-based hydrolysates showed high bioactivity and potential for use in serum-free media. • Enhanced galactosylation and sialylation was shown for some of the Mabs tested.

Gouy phase shift measurement using interferometric second-harmonic generation.

We report on a simple way to directly measure the Gouy phase shift of a strongly focused laser beam. This is accomplished by using a recent technique, namely, interferometric second-harmonic generation. We expect that this method will be of interest in a wide range of research fields, from high-harmonic and attosecond pulse generation to femtochemistry and nonlinear microscopy.

Mammalian Glucose Transporter Activity Is Dependent upon Anionic and Conical Phospholipids.

The regulated movement of glucose across mammalian cell membranes is mediated by facilitative glucose transporters (GLUTs) embedded in lipid bilayers. Despite the known importance of phospholipids in regulating protein structure and activity, the lipid-induced effects on the GLUTs remain poorly understood. We systematically examined the effects of physiologically relevant phospholipids on glucose transport in liposomes containing purified GLUT4 and GLUT3. The anionic phospholipids, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, were found to be essential for transporter function by activating it and stabilizing its structure. Conical lipids, phosphatidylethanolamine and diacylglycerol, enhanced transporter activity up to 3-fold in the presence of anionic phospholipids but did not stabilize protein structure. Kinetic analyses revealed that both lipids increase the kcat of transport without changing the Km values. These results allowed us to elucidate the activation of GLUT by plasma membrane phospholipids and to extend the field of membrane protein-lipid interactions to the family of structurally and functionally related human solute carriers.

Chronic Infection by Mucoid Pseudomonas aeruginosa Associated with Dysregulation in T-Cell Immunity to Outer Membrane Porin F.

RATIONALE: Pseudomonas aeruginosa (PA) is an environmental pathogen that commonly infects individuals with cystic fibrosis (CF) and non-CF bronchiectasis, impacting morbidity and mortality. To understand the pathobiology of interactions between the bacterium and host adaptive immunity and to inform rational vaccine design, it is important to understand the adaptive immune correlates of disease. OBJECTIVES: To characterize T-cell immunity to the PA antigen outer membrane porin F (OprF) by analyzing immunodominant epitopes in relation to infection status. METHODS: Patients with non-CF bronchiectasis were stratified by frequency of PA isolation. T-cell IFN-γ immunity to OprF and its immunodominant epitopes was characterized. Patterns of human leukocyte antigen (HLA) restriction of immunodominant epitopes were defined using HLA class II transgenic mice. Immunity was characterized with respect to cytokine and chemokine secretion, antibody response, and T-cell activation transcripts. MEASUREMENTS AND MAIN RESULTS: Patients were stratified according to whether PA was never, sometimes (<50%), or frequently (≥50%) isolated from sputum. Patients with frequent PA sputum-positive isolates were more likely to be infected by mucoid PA, and they showed a narrow T-cell epitope response and a relative reduction in Th1 polarizing transcription factors but enhanced immunity with respect to antibody production, innate cytokines, and chemokines. CONCLUSIONS: We have defined the immunodominant, HLA-restricted T-cell epitopes of OprF. Our observation that chronic infection is associated with a response of narrowed specificity, despite strong innate and antibody immunity, may help to explain susceptibility in these individuals and pave the way for better vaccine design to achieve protective immunity.

Nanomechanical mapping of hydrated rat tail tendon collagen I fibrils.

Collagen fibrils play an important role in the human body, providing tensile strength to connective tissues. These fibrils are characterized by a banding pattern with a D-period of 67 nm. The proposed origin of the D-period is the internal staggering of tropocollagen molecules within the fibril, leading to gap and overlap regions and a corresponding periodic density fluctuation. Using an atomic force microscope high-resolution modulus maps of collagen fibril segments, up to 80 µm in length, were acquired at indentation speeds around 10(5) nm/s. The maps revealed a periodic modulation corresponding to the D-period as well as previously undocumented micrometer scale fluctuations. Further analysis revealed a 4/5, gap/overlap, ratio in the measured modulus providing further support for the quarter-staggered model of collagen fibril axial structure. The modulus values obtained at indentation speeds around 10(5) nm/s are significantly larger than those previously reported. Probing the effect of indentation speed over four decades reveals two distinct logarithmic regimes of the measured modulus and point to the existence of a characteristic molecular relaxation time around 0.1 ms. Furthermore, collagen fibrils exposed to temperatures between 50 and 62°C and cooled back to room temperature show a sharp decrease in modulus and a sharp increase in fibril diameter. This is also associated with a disappearance of the D-period and the appearance of twisted subfibrils with a pitch in the micrometer range. Based on all these data and a similar behavior observed for cross-linked polymer networks below the glass transition temperature, we propose that collagen I fibrils may be in a glassy state while hydrated.

High-Throughput Production and Optimization of Membrane Proteins After Expression in Mammalian Cells.

High-quality protein samples are an essential requirement of any structural biology experiment. However, producing high-quality protein samples, especially for membrane proteins, is iterative and time-consuming. Membrane protein structural biology remains challenging due to low protein yields and high levels of instability especially when membrane proteins are removed from their native environments. Overcoming the twin problems of compositional and conformational instability requires an understanding of protein size, thermostability, and sample heterogeneity, while a parallelized approach enables multiple conditions to be analyzed simultaneously. We present a method that couples the high-throughput cloning of membrane protein constructs with the transient expression of membrane proteins in human embryonic kidney (HEK) cells and rapid identification of the most suitable conditions for subsequent structural biology applications. This rapid screening method is used routinely in the Membrane Protein Laboratory at Diamond Light Source to identify the most successful protein constructs and conditions while excluding those that will not work. The 96-well format is easily adaptable to enable the screening of constructs, pH, salts, encapsulation agents, and other additives such as lipids.

Structural Biology and Structure-Function Relationships of Membrane Proteins.

To understand the biological complexity of life, one needs to investigate how biomolecules behave and interact with each other at a molecular level [...].

The high-throughput production of membrane proteins.

Membrane proteins, found at the junctions between the outside world and the inner workings of the cell, play important roles in human disease and are used as biosensors. More than half of all therapeutics directly affect membrane protein function while nanopores enable DNA sequencing. The structural and functional characterisation of membrane proteins is therefore crucial. However, low levels of naturally abundant protein and the hydrophobic nature of membrane proteins makes production difficult. To maximise success, high-throughput strategies were developed that rely upon simple screens to identify successful constructs and rapidly exclude those unlikely to work. Parameters that affect production such as expression host, membrane protein origin, expression vector, fusion-tags, encapsulation reagent and solvent composition are screened in parallel. In this way, constructs with divergent requirements can be produced for a variety of structural applications. As structural techniques advance, sample requirements will change. Single-particle cryo-electron microscopy requires less protein than crystallography and as cryo-electron tomography and time-resolved serial crystallography are developed new sample production requirements will evolve. Here we discuss different methods used for the high-throughput production of membrane proteins for structural biology.

Transient Transfection and Expression of Eukaryotic Membrane Proteins in Expi293F Cells and Their Screening on a Small Scale: Application for Structural Studies.

Cancers, neurodegenerative and infectious diseases remain some of the leading causes of deaths worldwide. The structure-guided drug design is essential to advance drug development for these important diseases. One of the key challenges in the structure determination workflow is the production of eukaryotic membrane proteins (drug targets) of high quality. A number of expression systems have been developed for the production of eukaryotic membrane proteins. In this chapter, an optimized detailed protocol for transient transfection and expression of eukaryotic membrane proteins in Expi293F cells is presented. Testing expression and purification on a small scale allow optimizing conditions for sample preparation for downstream structural (cryo-EM) elucidation.

The structural basis of fatty acid elongation by the ELOVL elongases.

Very long chain fatty acids (VLCFAs) are essential building blocks for the synthesis of ceramides and sphingolipids. The first step in the fatty acid elongation cycle is catalyzed by the 3-keto acyl-coenzyme A (CoA) synthases (in mammals, ELOVL elongases). Although ELOVLs are implicated in common diseases, including insulin resistance, hepatic steatosis and Parkinson's, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which comprises an inverted transmembrane barrel surrounding a 35-Å long tunnel containing a covalently attached product analogue. The structure reveals the substrate-binding sites in the narrow tunnel and an active site deep in the membrane. We demonstrate that chain elongation proceeds via an acyl-enzyme intermediate involving the second histidine in the canonical HxxHH motif. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate, such as X-linked adrenoleukodystrophy.

Changes in Membrane Protein Structural Biology.

Membrane proteins are essential components of many biochemical processes and are important pharmaceutical targets. Membrane protein structural biology provides the molecular rationale for these biochemical process as well as being a highly useful tool for drug discovery. Unfortunately, membrane protein structural biology is a difficult area of study due to low protein yields and high levels of instability especially when membrane proteins are removed from their native environments. Despite this instability, membrane protein structural biology has made great leaps over the last fifteen years. Today, the landscape is almost unrecognisable. The numbers of available atomic resolution structures have increased 10-fold though advances in crystallography and more recently by cryo-electron microscopy. These advances in structural biology were achieved through the efforts of many researchers around the world as well as initiatives such as the Membrane Protein Laboratory (MPL) at Diamond Light Source. The MPL has helped, provided access to and contributed to advances in protein production, sample preparation and data collection. Together, these advances have enabled higher resolution structures, from less material, at a greater rate, from a more diverse range of membrane protein targets. Despite this success, significant challenges remain. Here, we review the progress made and highlight current and future challenges that will be overcome.

Mammalian cell culture for production of recombinant proteins: A review of the critical steps in their biomanufacturing.

The manufacturing of recombinant protein is traditionally undertaken in mammalian cell culture. Today, speed, cost and safety are the primary considerations for process improvements in both upstream and downstream manufacturing. Leaders in the biopharmaceutical industry are striving for continuous improvements to increase throughput, lower costs and produce safer more efficacious drugs. This can be achieved through advances in cell line engineering, process development of cell culture, development of chemically defined media and increased emphasis on product characterization. In the first part, this review provides a historical perspective on approved biotherapeutics by regulatory bodies which pave the way for next-generation products (including gene therapy). In the second part, it focuses on the application of in vitro and in vivo cell line engineering approaches, modern process development improvements including continuous manufacturing, recent developments in media formulation, and improvements in critical quality attribute determinations for products produced predominantly in mammalian cells.

The application of dispersive liquid-liquid microextraction in the analyses of the fatty acid profile in bovine milk in response to changes in body condition score.

Dispersive liquid-liquid microextraction (DLLME) was used prior to gas chromatography flame ionization detection (GC-FID) for the extraction of five fatty acids from milk taken from cows with different body condition scores. Optimum extraction conditions were: 300 µL of chloroform (extraction solvent), and 1 mL methanol (dispersive solvent). The procedure was optimised using Design of Experiments (DoE). The analytes were separated on a GC capillary column containing a polyethylene glycol stationary phase (15 m × 0.53 mm × 1.2 µm). Enrichment factors were in the range of 8-15 and limit of detection (LOD) was 0.04 µg/mL. Calibration graphs showed good linearity with coefficients of determination higher than 0.994% and relative standard deviations lower than 7%. This method provided a simple and rapid derivatisation and extraction method for the determination of fatty acids in bovine milk. It showed that there was a significant difference in the palmitic acid content of milk from cows that had an optimum body condition score (10.85 mg/mL) compared to cows that had a high body condition score (5.73 mg/mL).

Effect of seaweed supplementation on tocopherol concentrations in bovine milk using dispersive liquid-liquid microextraction.

A dispersive liquid-liquid microextraction (DLLME) method, combined with HPLC-UV detection, was developed for the extraction and preconcentration of δ-tocopherol from bovine milk. This method was used to study the effect of supplementing cow feed with the seaweed Ascophyllum nodosum on vitamin content in milk. The optimal experimental conditions were determined: 200 µL of chloroform (extraction solvent), 1.0 mL of ethanol (dispersive solvent), 5 mL of water (aqueous phase). Under these optimal conditions the DLLME method provided linearity in the range 0.01 µg/mL to 8 µg/mL with R2 values of 0.998. Limit of detection (LOD) was 0.01 µg/mL, while the enrichment factor was 89. Cow feed that was supplemented with Ascophyllum nodosum was shown to increase δ-tocopherol levels from 3.82 µg/mL to 5.96 µg/mL.

Combining tensile testing and structural analysis at the single collagen fibril level.

Tensile testing to failure followed by imaging is a simple way of studying the structure-function relationship of connective tissues such as skin, tendon, and ligament. However, interpretation of these datasets is complex due to the hierarchical structures of the tissues spanning six or more orders of magnitude in length scale. Here we present a dataset obtained through the same scheme at the single collagen fibril level, the fundamental tensile element of load-bearing tissues. Tensile testing was performed on fibrils extracted from two types of bovine tendons, adsorbed on a glass surface and glued at both ends. An atomic force microscope (AFM) was used to pull fibrils to failure in bowstring geometry. The broken fibrils were then imaged by AFM for morphological characterization, by second harmonic generation microscopy to assess changes to molecular packing, and by fluorescence microscopy after incubation with a peptide probe that binds specifically to denatured collagen molecules. This dataset linking stress-strain curves to post-failure molecular changes is useful for researchers modelling or designing functional protein materials.

In tendons, differing physiological requirements lead to functionally distinct nanostructures.

The collagen-based tissues of animals are hierarchical structures: even tendon, the simplest collagenous tissue, has seven to eight levels of hierarchy. Tailoring tissue structure to match physiological function can occur at many different levels. We wanted to know if the control of tissue architecture to achieve function extends down to the nanoscale level of the individual, cable-like collagen fibrils. Using tendons from young adult bovine forelimbs, we performed stress-strain experiments on single collagen fibrils extracted from tendons with positional function, and tendons with energy storing function. Collagen fibrils from the two tendon types, which have known differences in intermolecular crosslinking, showed numerous differences in their responses to elongation. Unlike those from positional tendons, fibrils from energy storing tendons showed high strain stiffening and resistance to disruption in both molecular packing and conformation, helping to explain how these high stress tissues withstand millions of loading cycles with little reparative remodeling. Functional differences in load-bearing tissues are accompanied by important differences in nanoscale collagen fibril structure.

Bowstring Stretching and Quantitative Imaging of Single Collagen Fibrils via Atomic Force Microscopy.

Collagen is the primary structural protein in animals. Serving as nanoscale biological ropes, collagen fibrils are responsible for providing strength to a variety of connective tissues such as tendon, skin, and bone. Understanding structure-function relationships in collagenous tissues requires the ability to conduct a variety of mechanical experiments on single collagen fibrils. Though significant advances have been made, certain tests are not possible using the techniques currently available. In this report we present a new atomic force microscopy (AFM) based method for tensile manipulation and subsequent nanoscale structural assessment of single collagen fibrils. While the method documented here cannot currently capture force data during loading, it offers the great advantage of allowing structural assessment after subrupture loading. To demonstrate the utility of this technique, we describe the results of 23 tensile experiments in which collagen fibrils were loaded to varying levels of strain and subsequently imaged in both the hydrated and dehydrated states. We show that following a dehydration-rehydration cycle (necessary for sample preparation), fibrils experience an increase in height and decrease in radial modulus in response to one loading-unloading cycle to strain <5%. This change is not altered by a second cycle to strain >5%. In fibril segments that ruptured during their second loading cycle, we show that the fibril structure is affected away from the rupture site in the form of discrete permanent deformations. By comparing the severity of select damage sites in both hydrated and dehydrated conditions, we demonstrate that dehydration masks damage features, leading to an underestimate of the degree of structural disruption. Overall, the method shows promise as a powerful tool for the investigation of structure-function relationships in nanoscale fibrous materials.