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bioRxiv2018

Stalk-mediated communication in the dynein motor domain

The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer-binding, and mechanical elements. Such communication is particularly complex and not well understood in the dynein motor, an ATPase that is comprised of a ring of six AAA domains, a large mechanical element (linker) spanning over the ring, and a microtubule-binding domain (MTBD) that is separated from the AAA ring by a ~135 [A] coiled-coil stalk. We identified mutations in the stalk that disrupt directional motion, have microtubule-independent hyperactive ATPase activity, and nucleotide-independent low affinity for microtubules. Cryo-electron microscopy structures of a mutant that uncouples ATPase activity from directional movement reveal that nucleotide-dependent conformational changes occur normally in one half of the AAA ring, but are disrupted in the other half. The large-scale linker conformational change observed in the wild-type protein is also inhibited, revealing that this conformational change is not required for ATP hydrolysis. These results demonstrate an essential role of the stalk in regulating motor activity and coupling conformational changes across the two halves of the AAA ring.

biochemistry↗

bioRxiv2018

Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides

We used electron cryo-micrographs to reconstruct actin filaments with bound AMPPNP ({beta},{gamma}-imidoadenosine 5-triphosphate, an ATP analog), ADP-Pi (ADP with inorganic phosphate) or ADP to resolutions of 3.4 [A], 3.4 [A] and 3.6 [A]. Subunits in the three filaments have nearly identical backbone conformations, so assembly rather than ATP hydrolysis or phosphate dissociation is responsible for their flattened conformation in filaments. Polymerization increases the rate of ATP hydrolysis by changing the conformations of the three ATP phosphates and the side chains of Gln137 and His161 in the active site. Flattening also promotes interactions along both the long-pitch and short-pitch helices. In particular, conformational changes in subdomain 3 open up favorable interactions with the DNase-I binding loop in subdomain 2 of the adjacent subunit. Subunits at the barbed end of the filament are likely to be in this favorable conformation, while monomers are not. This difference explains why filaments grow faster at the barbed end than the pointed end. Loss of hydrogen bonds after phosphate dissociation may account for the greater flexibility of ADP-actin filaments.\n\nSignificance StatementActin filaments comprise a major part of the cytoskeleton of eukaryotic cells and serve as tracks for myosin motor proteins. The filaments assemble from actin monomers with a bound ATP. After polymerization, actin rapidly hydrolyzes the bound ATP and slowly dissociates the {gamma}-phosphate. ADP-actin filaments then disassemble to recycle the subunits. Understanding how actin filaments assemble, disassemble and interact with numerous regulatory proteins depends on knowing the structure of the filament. High quality structures of ADP-actin filaments were available, but not of filaments with bound ATP- or with ADP and phosphate. We determined structures of actin filaments with bound AMPPNP (a slowly hydrolyzed ATP analog), ADP and phosphate and ADP by cryo-electron microscopy. These structures show how conformational changes during actin assembly promote ATP hydrolysis and faster growth at one end of the filament than the other.

biochemistry↗

bioRxiv2018

The histone chaperone FACT modulates nucleosome structure by tethering its components

Human FACT (hFACT) is a conserved histone chaperone that was originally described as a transcription elongation factor with potential nucleosome assembly functions. Here we show that FACT facilitates tetrasome assembly and H2A-H2B deposition to form hexasomes and nucleosomes. In the process, FACT tethers components of the nucleosome through interactions with H2A-H2B, resulting in a defined intermediate complex comprised of FACT, a histone hexamer and DNA. Free DNA extending from the tetrasome then competes FACT off H2A-H2B, thereby promoting hexasome and nucleosome formation. Our studies provide mechanistic insight into how FACT may stabilize partial nucleosome structures during transcription or nucleosome assembly, seemingly facilitating nucleosome disassembly and nucleosome assembly.

biochemistry↗

bioRxiv2018

Hydrogen-deuterium exchange coupled to top- and middle-down mass spectrometry enables high-resolution measurements of histone tail dynamics before and after nucleosome assembly

Until recently, a major limitation of hydrogen deuterium exchange mass spectrometry (HDX-MS) was that resolution of deuterium localization information was limited to the length of the peptide generated during proteolysis. Recently, however, it has been demonstrated that electron transfer dissociation (ETD) allows for preservation of deuterium label in the gas phase and therefore can be used to obtain more resolved information. To date, this technology has remained mostly limited to single, small, already well-characterized model proteins. Here, we optimize, expand, and adapt HDX-MS/MS capabilities to accommodate histone and nucleosomal complexes on top-down (TD) HDX-MS/MS and middle-down (MD) HDX-MS/MS platforms and demonstrate that near site-specific resolution of deuterium localization can be obtained with high reproducibility. We are able to study histone tail dynamics in unprecedented detail, which have evaded rigorous analysis by traditional structural biology techniques for decades, revealing important novel insights into chromatin biology. This work represents the first heterogeneous protein complex and protein-DNA complex to be analyzed by TD- and MD-HDX-MS/MS, respectively. Together, the results of these studies highlight the versatility, reliability, and reproducibility of ETD-based HDX-MS/MS methodology to interrogate large protein and protein/DNA complexes.

biochemistry↗

bioRxiv2018

Spc110 N-Terminal Domains Act Independently to Mediate Stable γ-Tubulin Small Complex Binding and γ-Tubulin Ring Complex Assembly

Microtubule (MT) nucleation in vivo is regulated by the {gamma}-tubulin ring complex ({gamma}TuRC), an approximately 2-megadalton complex conserved from yeast to humans. In Saccharomyces cerevisiae, {gamma}TuRC assembly is a key point of regulation over the MT cytoskeleton. Budding yeast {gamma}TuRC is composed of seven {gamma}-tubulin small complex ({gamma}TuSC) subassemblies which associate helically to form a template from which microtubules grow. This assembly process requires higher-order oligomers of the coiled-coil protein Spc110 to bind multiple {gamma}TuSCs, thereby stabilizing the otherwise low-affinity interface between {gamma}TuSCs. While Spc110 oligomerization is critical, its N-terminal domain (NTD) also plays a role that is poorly understood both functionally and structurally. In this work, we sought a mechanistic understanding of Spc110 NTD using a combination of structural and biochemical analyses. Through crosslinking-mass spectrometry (XL-MS), we determined that a segment of Spc110 coiled-coil is a major point of contact with {gamma}TuSC. We determined the structure of this coiled-coil segment by X-ray crystallography and used it in combination with our XL-MS dataset to generate an integrative structural model of the {gamma}TuSC-Spc110 complex. This structural model, in combination with biochemical analyses of Spc110 heterodimers lacking one NTD, suggests that the two NTDs within an Spc110 dimer act independently, one stabilizing association between Spc110 and {gamma}TuSC and the other stabilizing the interface between adjacent {gamma}TuSCs.

biochemistry↗

bioRxiv2018

Structural basis for transfer RNA mimicry by a bacterial Y RNA

Noncoding Y RNAs are present in both animal cells and many bacteria. In all species examined, Y RNAs tether the Ro60 protein to an effector protein to perform various cellular functions. For example, in the bacterium Deinococcus radiodurans, Y RNA tethers Ro60 to the exoribonuclease polynucleotide phosphorylase, specializing this nuclease for structured RNA degradation. Recently, a new Y RNA subfamily was identified in bacteria. Bioinformatic analyses of these YrlA (Y RNA-like A) RNAs predict that the effector-binding domain resembles tRNA. We present the structure of this domain, the overall folding of which is strikingly similar to canonical tRNAs. The tertiary interactions that are responsible for stabilizing tRNA are present in YrlA, making it a close tRNA mimic. However, YrlA lacks a free CCA end and contains a kink in the stem corresponding to the anticodon stem. Since nucleotides in the D and T stems are conserved among YrlAs, they may be an interaction site for an unknown factor. Our experiments identify YrlA RNAs as a new class of tRNA mimics.

biochemistry↗

bioRxiv2018

Cryo-EM structures reveal dynamic interplay of nascent chain-processing factors on the ribosome

During protein biosynthesis in bacteria, one of the earliest phenomena that a nascent polypeptide chain experiences is the co-translational enzymatic processing. The event includes two enzymatic pathways, deformylation of the N-terminal methionine followed by methionine excision catalyzed by peptide deformylase (PDF) and methionine aminopeptidase (MetAP). The ribosome tunnel exit serves as the podium for recruiting proteins involved in maturation processes of the nascent chain. During the process, the emerging nascent protein likely remains shielded by the chaperone trigger factor (TF).\n\nHere, we present the first cryo-EM structures of E. coli ribosome in complex with the nascent chain processing proteins. The structures reveal overlapping binding sites for PDF and MetAP when they bind individually at the tunnel exit site, where proteins L22 and L32 are identified as primary anchoring sites for both proteins. Interestingly however, MetAP has a remarkable ability of repositioning itself to adjacent locations in the presence of PDF and TF at the tunnel exit. Thus, our results disclose an unexpected scanning mechanism that MetAP adopts for context-specific ribosome association.

biochemistry↗

bioRxiv2018

Substrate binding allosterically relieves autoinhibition of the TRIB1 pseudokinase

One Sentence SummarySubstrate binding to Tribbles-homolog 1 (TRIB1) pseudokinase induces allosteric changes that allow formation of a complex with the COP1 ubiquitin ligase.\n\nAbstractThe Tribbles family of pseudokinases recruit substrates to the COP1 ubiquitin ligase for ubiquitination. CCAAT-enhancer binding protein (C/EBP) family transcription factors are crucial Tribbles substrates in adipocyte and myeloid development. Here we show that the TRIB1 pseudokinase can recruit various C/EBP family members, with binding of C/EBP{beta} attenuated by phosphorylation. To explain the mechanism of substrate recruitment, we solved the crystal structure of TRIB1 in complex with C/EBP. TRIB1 undergoes a significant conformational change relative to its substrate-free structure, to bind C/EBP in a pseudo-substrate-like manner. Crucially, substrate binding triggers allosteric changes that link substrate recruitment to COP1 binding, which is consistent with molecular dynamics and biochemical studies. These findings offer a view of pseudokinase regulation with striking parallels to bona fide kinase regulation-- via the activation loop and C-helix--and raise the possibility of small molecules targeting either the activation loop-in, or loop-out, conformations of Tribbles pseudokinases.

biochemistry↗

bioRxiv2018

Structural basis for the RNA-guided ribonuclease activity of CRISPR-Cas13d

CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function, we resolved cryo-electron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 [A] resolution, respectively. Furthermore, a 6.5 [A] reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, explain the compact molecular architecture of Cas13d and provide insights into the structural transitions required for enzyme activation. Our comprehensive analysis of Cas13d in diverse enzymatic states facilitated site-specific truncations for minimal size and delineates a blueprint for improving biomolecular applications of RNA targeting.

biochemistry↗

bioRxiv2018

Synthesis of oxyfunctionalized NSAID metabolites by microbial biocatalysts

The synthesis of valuable metabolites and degradation intermediates of drugs, like non-steroidal anti-inflammatory drugs (NSAIDs), are substantially for toxicological and environmental studies, but efficient synthesis strategies and the metabolite availability are still challenging aspects. To overcome these bottlenecks filamentous fungi as microbial biocatalysts were applied. Different NSAIDs like diclofenac, ibuprofen, naproxen and mefenamic acid could be oxyfunctionalized to produce human metabolites in isolated yields of up to 99% using 1 g L-1 of substrate. Thereby the biotransformations using Beauveria bassiana, Clitocybe nebularis or Mucor hiemalis surpass previous reported chemical, microbial and P450-based routes in terms of efficiency. In addition to different hydroxylated compounds of diclofenac, a novel metabolite, 3,4-dihydroxydiclofenac, has been catalyzed by B. bassiana and the responsible P450s were identified by proteome analysis. The applied filamentous fungi present an interesting alternative, microbial biocatalysts platform for the production of valuable oxyfunctionalized drug metabolites.\n\nImportanceThe occurrence of pharmaceutically active compounds, such as diclofenac and its metabolites, in the environment, in particular in aquatic systems, is of increasing concern because of the increased application of drugs. Standards of putative metabolites are therefore necessary for environmental studies. Moreover, pharmaceutical research and development requires assessment of the bioavailability, toxicity and metabolic fate of potential new drugs to ensure its safety for users and the environment. Since most of the reactions in the early pharmacokinetics of drugs are oxyfunctionalizations catalysed by P450s, oxyfunctionalized metabolites are of major interest. However, to assess these metabolites chemical synthesis often suffer from multistep reactions, toxic substances, polluting conditions and achieve only low regioselectivity. Biocatalysis can contribute to this by using microbial cell factories. The significance of our research is to complement or even exceed synthetic methods for the production of oxyfunctionalized drug metabolites.

biochemistry↗

bioRxiv2018

Analyzing Enzyme Kinetic Data Using the Powerful Statistical Capabilities of R

We describe a powerful tool for enzymologists to use for typical non-linear fitting of common equations in enzyme kinetics using the statistical program R. Enzyme kinetics is a powerful tool for understanding enzyme catalysis, regulation, and inhibition but tools to perform the analysis have limitations. Software to perform the necessary nonlinear analysis may be proprietary, expensive or difficult to use, especially for a beginner. The statistical program R is ideally suited to analyzing enzyme kinetic data; it is free in two respects: there is no cost and there is freedom to distribute and modify. It is also robust, powerful and widely used in other fields of biology. In this paper we introduce the program R to enzymologists who want to analyze their data but are unfamiliar with R or similar command line statistical analysis programs. Data are inputted and examples of different non-linear models are fitted. Results are extracted and plots are generated to assist judging the goodness of fit. The instructions will allow users to create their own modifications to adapt the protocol to their own experiments. Because of the use of scripts, a method can be modified and used to analyze different datasets in less than one hour.

biochemistry↗

bioRxiv2018

Identification, structure-activity relationship and in silico molecular docking analyses of five novel angiotensin I-converting enzyme (ACE)-inhibitory peptides from stone fish hydrolysates

Stone fish is an under-utilized sea cucumber with many health benefits. Hydrolysates with strong ACE-inhibitory effects were generated from stone fish protein under the optimum conditions of hydrolysis using bromelain and fractionated based on hydrophobicity and isoelectric properties of the constituent peptides. Five novel peptide sequences with molecular weight (mw) < 1000 daltons (Da) were identified using LC-MS/MS. The peptides including ALGPQFY (794.44 Da), KVPPKA (638.88 Da), LAPPTM (628.85 Da), EVLIQ (600.77 Da) and EHPVL (593.74 Da) were evaluated for ACE-inhibitory activity and showed IC50 values of 0.012 mM, 0.980 mM, 1.31 mM, 1.44 mM and 1.68 mM, respectively. The ACE-inhibitory effects of the peptides were further verified using molecular docking study. The docking results demonstrated that the peptides exhibit their effect mainly via hydrogen and electrostatic bond interactions with ACE. These findings provide evidence about stone fish as a valuable source of raw materials for the manufacture of antihypertensive peptides that can be incorporated to enhance therapeutic relevance and commercial significance of formulated functional foods.

biochemistry↗

bioRxiv2018

Elucidating the roles of Alzheimer disease-associated proteases and the signal-peptide peptidase-like 3 (SPPL3) in the shedding of glycosyltransferases

The Golgi resident glycosyltransferases (GTs) are membrane-bound glycoproteins but are frequently found as soluble proteins in biological fluids where their function remains largely unknown. Previous studies have established that the release of these proteins involved Alzheimer disease-associated proteases such as {beta}-secretases (BACE1 and BACE2) and the intramembrane-cleaving aspartyl proteases Presenilins 1 and 2. Recent studies have involved another intramembrane-cleaving enzyme, the signal peptide peptidese-like-3 (SPPL3). Except for the latter, the two former studies mostly addressed particular cases of GTs, namely ST6Gal-I (BACEs) or GnT-V (Presenilins). Therefore the question still remains as which of these secretases is truly responsible for the cleavage and secretion of GTs. We herein combined the 3 proteases in a single study with respect to their abilities to release 3 families of GTs encompassing three N-acetylglucosaminyltransferases, two fucosyltransferases and two sialyltransferases. Green fluorescent protein (gfp)-fused versions of these GTs were virally transduced in mouse embryonic fibroblasts devoid of BACEs, Presenilins or SPPL3. We found that neither BACE nor Presenilins are involved in the shedding of these glycosyltransferases, while SPPL3 was involved in the cleavage and release of some but not all GTs. Notably, the {gamma}- secretase inhibitor DFK-167 was the only molecule capable of significantly decreasing glycosyltransferase secretion, suggesting the involvement of {gamma}-secretase(s), yet different from Presenilins but comprising SPPL3 among other proteases still to be identified. Using confocal microscopy, we show that SPPL3 selectivity towards GTs relays not only on sequence specificity but also depends on how GTs distribute in the cell with respect SPPL3 during their cycling within and outside the Golgi.

biochemistry↗

bioRxiv2018

Binding specificities of human RNA binding proteins towards structured and linear RNA sequences

Sequence specific RNA-binding proteins (RBPs) control many important processes affecting gene expression. They regulate RNA metabolism at multiple levels, by affecting splicing of nascent transcripts, RNA folding, base modification, transport, localization, translation and stability. Despite their central role in most aspects of RNA metabolism and function, most RBP binding specificities remain unknown or incompletely defined. To address this, we have assembled a genome-scale collection of RBPs and their RNA binding domains (RBDs), and assessed their specificities using high throughput RNA-SELEX (HTR-SELEX). Approximately 70% of RBPs for which we obtained a motif bound to short linear sequences, whereas ~30% preferred structured motifs folding into stem-loops. We also found that many RBPs can bind to multiple distinctly different motifs. Analysis of the matches of the motifs in human genomic sequences suggested novel roles for many RBPs. We found that three cytoplasmic proteins, ZC3H12A, ZC3H12B and ZC3H12C bound to motifs resembling the splice donor sequence, suggesting that these proteins are involved in degradation of cytoplasmic viral and/or unspliced transcripts. Surprisingly, structural analysis revealed that the RNA motif was not bound by the conventional C3H1 RNA-binding domain of ZC3H12B. Instead, the RNA motif was bound by the ZC3H12Bs PilT N-terminus (PIN) RNase domain, revealing a potential mechanism by which unconventional RNA binding domains containing active sites or molecule-binding pockets could interact with short, structured RNA molecules. Our collection containing 145 high resolution binding specificity models for 86 RBPs is the largest systematic resource for the analysis of human RBPs, and will greatly facilitate future analysis of the various biological roles of this important class of proteins.

biochemistry↗

bioRxiv2018

Electrostatic repulsion causes anticooperative DNA binding between tumor suppressor ETS transcription factors and JUN-FOS at composite DNA sites

Many transcription factors regulate gene expression in a combinatorial fashion often by binding in close proximity on composite cis-regulatory DNA elements. Here we investigate the molecular basis by which ETS transcription factors bind with AP1 transcription factors JUN-FOS at composite DNA-binding sites. The ability to bind to DNA with JUN-FOS correlates with the phenotype of these proteins in prostate cancer: the oncogenic ERG and ETV1/4/5 subfamilies co-occupy ETS-AP1 sites with JUN-FOS in vitro, whereas JUN-FOS robustly inhibits DNA binding by the tumor suppressors EHF and SPDEF. EHF binds to ETS-AP1 DNA with tighter affinity than ERG in the absence of JUN-FOS, which may enable EHF to compete with ERG and JUN-FOS for binding to ETS-AP1 sites. Genome-wide mapping of EHF and ERG binding sites in a prostate epithelial cell line reveal that EHF is preferentially excluded from closely spaced ETS-AP1 DNA sequences. Structural modeling and mutational analyses indicate that adjacent positively-charged surfaces from EHF and JUN-FOS disfavor simultaneous DNA binding due to electrostatic repulsion. The conservation of positively charged residues on the JUN-FOS interface identified ELF1 as an additional ETS factor that exhibits anticooperative DNA binding, and we present evidence that ELF1 is frequently downregulated in prostate cancer. In summary, the divergence of electrostatic features of ETS factors at their JUN-FOS interface enables distinct binding events at ETS-AP1 DNA sequences. We propose that this mechanism can drive unique targeting of ETS transcription factors, thereby facilitating distinct transcriptional programs.

biochemistry↗

bioRxiv2018

Three enzymes and one substrate; regulation of flux through the glyoxylate shunt in the opportunistic pathogen, Pseudomonas aeruginosa.

The glyoxylate shunt bypasses the oxidative decarboxylation steps of the tricarboxylic acid (TCA) cycle, thereby conserving carbon skeletons for biosynthesis. The branchpoint between the TCA cycle and the glyoxylate shunt is therefore widely considered to be one of the most important junctions in the whole of microbial metabolism. In Escherichia coli, AceK-mediated phosphorylation and inactivation of the TCA cycle enzyme, isocitrate dehydrogenase (ICD), is necessary to redirect flux through the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL). In contrast, Mycobacterial species lack AceK and employ a phosphorylation-insensitive isocitrate dehydrogenase (IDH) at the branchpoint. Flux partitioning here is controlled \"rheostatically\" through cross-activation of IDH by the product of ICL activity, glyoxylate. However, the opportunistic human pathogen, Pseudomonas aeruginosa, expresses IDH, ICD, ICL and AceK. Here, we present the structure, kinetics and regulation of each branchpoint enzyme. We show that flux partitioning is coordinated through reciprocal regulation of the enzymes involved, beautifully linking carbon flux with the availability of key gluconeogenic precursors in a way that cannot be extrapolated from an understanding of the branchpoint enzymes in other organisms.

biochemistry↗

bioRxiv2018

Soft drinks and monogenetic diabetes: a study on the Wolfram syndrome 1 (Wfs1) deficient mouse model

In a modern society, the risk of developing type II diabetes and obesity may be linked to the increased consumption of carbohydrate-rich drinks. Several genes, including Wolfram Syndrome 1 (WFS1), have been reported to increase susceptibility for developing type II diabetes. In this study we aimed to investigate the effect of chronic consumption of carbohydrate-rich drinks on weight gain, overall consumption of liquids, glucose tolerance and liver metabolism in Wfs1-deficient mice. Wfs1-deficient and wild-type mice were divided into three groups that consumed regular Coca-Cola, 20% sucrose solution or water ad libitum as the only source of liquid. During the experiment, daily liquid consumption was determined. After 30 days, total weight gain of mice was calculated and glucose tolerance test was performed. The liver tissue was analysed by means of untargeted and targeted metabolomics using liquid chromatography-mass spectrometry. Weight gain was strongly affected by mouse genotype (p<0.001), their drink (p<0.001) and the interaction of both genotype and drink (p<0.001). Coca-Cola significantly increased liquid consumption in knock-out mice. There was an effect of the drink (p<0.001) and the interaction between the genotype and treatment (p=0.02) on blood glucose level while Coca-Cola and 20% sucrose solution exacerbated glucose intolerance in the knock-out mice. In untargeted metabolic profiling, the water consuming wild-type and heterozygous mice were found to be the most distinctive from the mice with all other genotype and drink combinations. Targeted analysis revealed interactions between the genotype and drink regarding to glycolysis and lipogenesis. In the wild-type animals, carbohydrate overload was alleviated by converting glucose to lipids. However, the same mechanism is not implemented in knock-out animals, as lipolysis and gluconeogenesis are upregulated by Wfs1 deficiency. In conclusion, our study demonstrates a significant interaction between the genotype and the drink when comparing wild-type and Wfs1 knock-out mice consuming soft drinks.

biochemistry↗

bioRxiv2018

Expression and purification of a functional heteromeric GABAA receptor for structural studies

The multi-subunit GABA-gated chloride channels of the Cys-loop receptor family, known as GABAA receptors, function as the primary gatekeepers of fast inhibitory neurotransmission in the central nervous system. In addition to their role in controlling synaptic tone, these receptors are the targets of a vast array of therapeutic compounds that potentiate channel gating. Importantly, functional activity and pharmacological efficacy of GABAA receptors is coupled directly to the subunit composition. However, the absence of high resolution structural information precludes an explicit determination of the molecular mechanism of ligand binding to ion channel gating and modulation. Efforts to obtain this data are hindered largely by the lack of heterologous expression and purification protocols for high expressing receptor constructs. To address this issue, we describe a unique approach to identify bona fide functional GABAA receptor subunit combinations by using the Xenopus oocyte as an expression host in combination with fluorescence detection size exclusion chromatography. The results demonstrate that formation of a defined pentameric species is dependent on subunit composition. Furthermore, receptor subunits can tolerate large truncations in non-conserved M3/M4 cytoplasmic loop, although removal of N-linked glycosylation sites is negatively correlated with expression level. Additionally, we report methods to improve GABAA receptor expression in mammalian cell culture that employ recombinant baculovirus transduction. From these methods we have identified a well-behaving minimal functional construct for the 1/{beta}1 GABAA receptor subtype that can be purified in milligram quantities while retaining high affinity agonist binding activity.

biochemistry↗