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Results for “Biochemistry”

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At least 1,153 records · Page 64Linked to original sources

bioRxiv2018

A practical method for efficient and optimal production of selenomethionine-labeled recombinant protein complexes in the insect cells

The use of Selenomethionine (SeMet) incorporated protein crystals for single or multiwavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X-ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in E. coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet-incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allows for incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60-90% compared to native protein expression.

biochemistry↗

bioRxiv2018

Structural Basis for Stereoselective Dehydration and Hydrogen-Bonding Catalysis by the SAM-Dependent Pericyclase LepI

LepI is an S-adenosylmethionine (SAM)-dependent pericyclase that catalyzes the formation of 2-pyridone natural product leporin C. Biochemical characterization showed LepI can catalyze the stereoselective dehydration to yield a reactive (E)-quinone methide which can undergo a bifurcating intramolecular Diels-Alder (IMDA) and hetero-Diels-Alder (HDA) cyclization from an ambimodal transition state, and a [3,3]-retro-Claisen rearrangement to recycle the IMDA product into leporin C. Here we solved the X-ray crystal structures of SAM-bound LepI, and in complex with a substrate analog, the product leporin C, and a retro-Claisen reaction transition-state analog to understand the structural basis for the multitude of reactions. Structural and mutational analysis revealed how Nature evolves a classic methyltransferase active site into one that can serve as a dehydratase and a multifunctional pericyclase. Catalysis of both sets of reactions employ His133 and Arg295, two active site residues that are not found in canonical methyltransferases. An alternative role of SAM, which is not found to be in direct contact of the substrate, is also proposed.

biochemistry↗

bioRxiv2018

Design and characterisation of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

The gene mutated in Huntingtons disease (HD) patients encodes the 348 kDa huntingtin (HTT) protein. The pathogenic HD CAG-expansion mutation causes a polyglutamine (polyQ) tract at the N-terminus of the HTT protein to expand above a critical threshold of ~35 glutamine residues. The effect of HD mutations on HTT is not well understood, in part due to difficulties in carrying out biochemical, biophysical and structural studies of this large protein. To facilitate such studies, we have generated expression constructs for the scalable production of HTT in multiple eukaryotic expression systems. Our set of HTT expression clones comprises both N and C-terminally FLAG-tagged HTT constructs with polyQ lengths representative of the general population, HD patients, juvenile HD patients as well as the more extreme polyQ expansions used in some HD tissue and animal models. These reagents yield milligram quantities of pure recombinant HTT protein, including many of the previously mapped posttranslational modifications. We have characterised both apo and HTT-HAP40 complex samples produced using this HD resource, demonstrating that this toolkit can be used to generate physiologically meaningful complexes of HTT. We demonstrate how these resources can produce sufficient material for protein-intensive experiments such as small angle X-ray scattering (SAXS), providing biochemical insight into HTT protein structure. The work outlined in this manuscript and the tools generated, lay a foundation for further biochemical and structural work on the HTT protein and its functional interactions with other biomolecules.

biochemistry↗

bioRxiv2018

Membrane curvature sensing of the lipid-anchored K-Ras small GTPases

Cell morphologies, defined by plasma membrane (PM) local curvature, change during mitogen-dependent function and pathology, such as growth, division and proliferation. The lipid-anchored Ras small GTPases are essential upstream regulators of the mitogen-activated protein kinases (MAPKs) cascades and play key roles in many pathological conditions, especially cancer. Ras signaling is mostly compartmentalized to the cell PM through the formation of nanometer-sized domains, termed as nanoclusters, and undergo selective lipid sorting for efficient effector recruitment and activation. Thus, Ras function might be sensitive to changing PM curvature, potentially regulating mechanosensing of mitogen signaling. We employed nanofabrication and super-resolution imaging and found that Ras functions respond to PM curvature modulations in an isoform specific manner: nanoclustering and signaling of the most oncogenically prevalent isoform K-Ras favor less curved PM, while those of another isoform H-Ras favor more curved PM. We then examined whether Ras membrane curvature sensing is mediated by lipid sorting. We found that anionic phospholipids sense changing PM curvature in distinct manners: phosphatidylserine (PS) localization shows preference for less curved membrane but phosphoinositol 4,5-bisphosphate (PIP2) localization favors more curved PM. Depletion of endogenous PS abolishes K-Ras PM curvature sensing. Exogenous PS addback and synthetic bilayer binding assays further show that only mixed-chain PS species, but not other PS species, mediate K-Ras curvature sensing. Taken together, the Ras proteolipid nano-assemblies on the PM act as relay stations to convert mechanical stimulations to mitogenic signaling circuits, thus a novel mechanism for cancer cell mechanotransduction

biochemistry↗

bioRxiv2018

Identification of three sequence motifs in the transcription termination factor Sen1 that mediate direct interactions with Nrd1

The Nrd1-Nab3-Sen1 (NNS) complex carries out the transcription termination of noncoding RNAs (ncRNAs) in yeast, although the detailed interactions among its subunits remain obscure. Here we have identified three sequence motifs in Sen1 that mediate direct interactions with the RNA polymerase II CTD interaction domain (CID) of Nrd1, determined the crystal structures of these Nrd1 interaction motifs (NIMs) bound to the CID, and characterized the interactions in vitro and in yeast.

biochemistry↗

bioRxiv2018

Unique unfoldase/aggregase activity of a molecular chaperone that dysregulates a translational elongation factor

The various chaperone activities of heat shock proteins contribute to ensuring cellular proteostasis. Here, we demonstrate the non-canonical unfoldase activity as an inherent functionality of the prokaryotic molecular chaperone Hsp33. The holding-inactive, reduced form of Hsp33 (RHsp33) strongly bound to the translational elongation factor, EF-Tu, and catalyzed the EF-Tu aggregation via evoking its aberrant folding, resulting in its susceptibility to proteolytic degradation by Lon. This interaction was critically mediated by the redox-switch domain of RHsp33 and the guanine nucleotide-binding domain of EF-Tu. The RHsp33-induced in vivo aggregation of EF-Tu upon heat shock was evident in a Lon-deficient strain and inhibited cell growth. Unlike wild-type Escherichia coli, the strain lacking both Hsp33 and Lon showed a non-reduced level of EF-Tu and diminished capability of counteracting heat shock. These findings suggest that the unique unfoldase/aggregase activity of Hsp33 potentially involved in protein turnover confers a cellular survival advantage under heat-stressed conditions.

biochemistry↗

bioRxiv2018

Upregulation of photosynthetic capacity and thylakoid membrane protein enhanced tolerance to heat stress in wucai (Brassica campestris L.)

The hot climate of southern China from late summer to early fall is one of the major factors limiting the yield and quality of wucai (Brassica campestris L.). Under high temperature stress, heat-tolerant cultivars presented moderate injury to the photosynthesic apparatus, less inhibition of photochemical activity, better osmotic adjustment and antioxidant defences capacity compared to heat-sensitive cultivars. To study the effects of high temperature on the growth and development of wucai, plants of WS-1 (heat-tolerant) and WS-6 (heat-sensitive) were exposed to four heat stress treatments in growth chambers for 3 days. Chloroplasts of two cultivars evaluated for photosynthetic characteristics, fatty acid composition and differentially expressed proteins of thylakoid membrane. The chlorophyll (Chl) content was markedly reduced by heat stress, inhibiting photochemical activity. However, larger decreases in growth and photosynthetic parameters [net photosynthetic rate (PN), stomatal conductance (GS), intercellular CO2 concentration (Ci), and leaf transpiration rate (E)] occurred under heat stress in WS-6, compared with WS-1. In addition, WS-6 showed an obviouse K point in O-J-I-P steps under extremely high temperature, which indicated OEC had been damaged. WS-1 showed higher of maximum quantum yield of primary PSII photochemistry (FV/FM), number of active reaction centres per cross section of PSII (RC/CSM), average absorbed photon flux per cross section of PSII (ABS/CSM), maximum trapped exciton flux per cross section of PSII (TR0/CSM), electron transport flux from QA to QB per cross section of PSII (ET0/ CSM) and performance index on absorption basis (PIABS) which indicated greater heat stability in terms of PSII function under higher temperature. Compared to WS-6, WS-1 showed higher membrane stability and photochemical efficiency, and greater increase of saturated fatty acids (SFA), especially palmitic acid under heat stress. WS-1 had higher recovery rate compared to WS-6 after 41{degrees}C heat stress treatment. Additionally, two-dimensional blue native/SDS-PAGE analysis of chloroplast was carried out to compare the differentially expressed proteins between two cultivars. We obtained seven major protein complexes included supercomplexes, PSI-LHCII/PSII monomer, PSII monomer, CP43 less PSII/ATP synthase, LHCII trimer, LHCII monomer and ATP synthase after first dimentional seperation in both cultivars after the first dimensional separation. Then ten differential membrane proteins included light-harvesting Chl a/b-binding (LHC) protein, ATP synthase subunit alpha, ATP synthase subunit beta, photosystem I P700 chlorophyll a apoprotein A2, photosystem II CP43 reaction center protein, photosystem II D2 protein and photosystem II\n\nOS have been found between WS-1 and WS-6. These differentially proteins in cellular membranes could contribute to the differential level of heat tolerance between two wucai cultivars. Our results demonstrated that the heat-tolerant cultivar WS-1 had a greater capacity for photosynthesis and membrane stability by upregulating proteins abundance including light harvesting (light-harvesting Chl a/b-binding protein), energy metabolism (ATPase), and proteins of PSII reaction center (D2, CP43) under heat stress.

biochemistry↗

bioRxiv2018

Structural Insights into Curli Amyloid Fibrils suggest Repurposing Anti-amyloid Compounds as Anti-biofilm Agents

Curli amyloid fibrils secreted by Enterobacteriaceae mediate host cell adhesion and contribute to biofilm formation, thereby promoting bacterial resistance to environmental stressors. Here, we present crystal structures of amyloid-forming segments from the major curlin subunit, CsgA, revealing steric zipper fibrils of tightly mated {beta}-sheets, demonstrating a structural link between curli and human pathological amyloids. We propose that these cross-{beta} segments structure the highly robust curli amyloid core. D-enantiomeric peptides, originally developed to interfere with Alzheimers disease-associated Amyloid-{beta}, inhibited CsgA fibrillation and reduced biofilm formation in Salmonella typhimurium. Moreover, CsgA fibrils cross-seeded fibrillation of Amyloid-{beta}, providing further support for the proposed structural resemblance and potential for cross-species amyloid interactions. In this study, we provide structural insights into curli formation, offer a novel strategy for disrupting amyloid-structured biofilms, and hypothesize on the formation of self-propagating prion-like species originating from a microbial source that could influence neurodegenerative diseases.\n\nSignificanceAtomic resolution structural insights into the biofilm-associated curli amyloid fibril secreted by Enterobacteriaceae revealed elements of fibrillar architecture conserved between bacterial and human amyloids. This inspired us to repurpose anti-amyloid drugs designed to target human pathological amyloids as a novel class of anti-biofilm agents. Moreover, the results provide a molecular basis for understanding interspecies cross-seeding of amyloids through the generation of prion-like agents by molecular mimicry. This raises concerns regarding human exposure to exogenous sources of amyloids, such as contaminated food and amyloid-secreting microbes. Overall, we provide a novel framework for investigating interspecies amyloid interactions at the molecular level and offer novel insights into mechanisms which may underlie the evolutionary and etiological relationships between the human and microbial amylomes.

biochemistry↗

bioRxiv2018

Broken symmetry in the human BK channel

Voltage-gated and ligand-modulated ion channels play critical roles in excitable cells. To understand the interplay among voltage-sensing, ligand-binding and channel opening, the structures of ion channels in various functional states need to be determined. Here, the \"random spherically constrained\" (RSC) single-particle cryo-EM method was employed to study the human large conductance voltage- and calcium-activated potassium (hBK or hSlo1) channels reconstituted into liposomes. The hBK structure has been determined at 3.5 [A] resolution in the absence of Ca2+. Instead of the common four-fold symmetry observed in ligand-modulated ion channels, a two-fold symmetry was observed in hBK. Two opposing subunits in the Ca2+ sensing gating ring rotate around the center of each subunit, which results in the movement of the assembly and flexible interfaces and Ca2+ binding sites. Despite the significant movement, the local conformation of the assembly interfaces and Ca2+ binding sites remains the same among the four subunits.

biochemistry↗

bioRxiv2018

Kinetic And Structural Parameters Governing Fic-Mediated Adenylylation/AMPylation of the Hsp70 chaperone, BiP/GRP78

Fic (filamentation induced by cAMP) proteins regulate diverse cell signaling events by post-translationally modifying their protein targets, predominantly by the addition of an AMP (adenosine monophosphate). This modification is called Fic-mediated Adenylylation or AMPylation. We previously reported that the human Fic protein, HYPE/FicD, is a novel regulator of the unfolded protein response (UPR) that maintains homeostasis in the endoplasmic reticulum (ER) in response to stress from misfolded proteins. Specifically, HYPE regulates UPR by adenylylating the ER chaperone, BiP/GRP78, which serves as a sentinel for UPR activation. Maintaining ER homeostasis is critical for determining cell fate, thus highlighting the importance of the HYPE-BiP interaction. Here, we study the kinetic and structural parameters that determine the HYPE-BiP interaction. By measuring the binding and kinetic efficiencies of HYPE in its activated (Adenylylation-competent) and wild type (de-AMPylation-competent) forms for BiP in its wild type and ATP-bound conformations, we determine that HYPE displays a nearly identical preference for the wild type and ATP-bound forms of BiP in vitro and preferentially de-AMPylates the wild type form of adenylylated BiP. We also show that AMPylation at BiPs Thr366 versus Thr518 sites differentially affect its ATPase activity, and that HYPE does not adenylylate UPR accessory proteins like J-protein ERdJ6. Using molecular docking models, we explain how HYPE is able to adenylylate Thr366 and Thr518 sites in vitro. While a physiological role for AMPylation at both the Thr366 and Thr518 sites has been reported, our molecular docking model supports Thr518 as the structurally preferred modification site. This is the first such analysis of the HYPE-BiP interaction and offers critical insights into substrate specificity and target recognition.

biochemistry↗

bioRxiv2018

Cryo-EM sample preparation method for extremely low concentration liposomes

Liposomes are widely used as delivery systems in pharmaceutical, cosmetics and food industries, as well as a system for structural and functional study of membrane proteins. To accurately characterize liposomes, cryo-Electron Microscopy (cryo-EM) has been employed as it is the most precise and direct method to determine liposome lamellarity, size, shape and ultrastructure. However, its use is limited by the number of liposomes that can be trapped in the thin layer of ice that spans holes in the perforated carbon film on EM grids. We report a long-incubation method for increasing the density of liposomes in holes. By increasing the incubation time, high liposome density was achieved even with extremely dilute (in the nanomolar range) liposome solutions. This long-incubation method has been successfully employed to study the structure of an ion channel reconstituted into liposomes. This method will also be useful for preparing other biological macromolecules / assemblies for structural studies using cryo-EM.

biochemistry↗

bioRxiv2018

cGAMP signaling controls a transient surface-associated lifestyle

A newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated with bacterial cyclic di-GMP signaling. This system provides a rare opportunity to study how signaling proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, the Hypr GGDEF in Geobacter sulfurreducens, specifically regulates cyclic GMP-AMP (3,3-cGAMP) levels in vivo to stimulate gene expression associated with metal reduction separate from electricity production. To reconcile these in vivo findings with prior in vitro results that showed GacA was promiscuous, we developed a full kinetic model combining experimental data and mathematical modeling to reveal mechanisms that contribute to in vivo specificity. A 1.4 [A]-resolution crystal structure of the Geobacter Hypr GGDEF domain was determined to understand the molecular basis for those mechanisms, including key cross-dimer interactions. Together these results demonstrate that specific signaling can result from a promiscuous enzyme.

biochemistry↗

bioRxiv2018

Novel strand exchange activity of the human PALB2 DNA Binding Domain and its critical role for DNA repair in cells

Breast cancer associated proteins 1 and 2 (BRCA1, -2) and partner and localizer of BRCA2 (PALB2) protein are tumor suppressors linked to a spectrum of malignancies, including breast cancer and Fanconi anemia. They stimulate RAD51 recombinase during homology-directed repair (HDR). Along with being a hub for a protein interaction network, PALB2 interacts with DNA. The mechanism of PALB2 DNA binding and its function are poorly understood. We identified a major DNA-binding site in PALB2, mutation of which reduces the RAD51 foci formation and the overall HDR efficiency in cells by 50%. PALB2 N-terminal DNA-binding domain (N-DBD) stimulates the RAD51 strand exchange reaction. Surprisingly, it promotes the strand exchange without RAD51. Moreover, N-DBD stimulates the inverse strand exchange and can use both DNA and RNA substrates. Our data reveal a versatile DNA interaction property of PALB2 and demonstrate a critical role of PALB2 DNA binding for chromosome repair in cells.

biochemistry↗

bioRxiv2018

Identifying the Biosynthetic Gene Cluster for Triacsins with an N-hydroxytriazene Moiety

Triacsins are a family of natural products containing an N-hydroxytriazene moiety not found in any other known secondary metabolites. Though many studies have examined the biological activity of triacsins in lipid metabolism, the biosynthesis of triacsins has remained unknown. Here, we report the identification of the triacsin biosynthetic gene cluster in Streptomyces aureofaciens ATCC 31442. Bioinformatic analysis of the gene cluster led to the discovery of the tacrolimus producer Streptomyces tsukubaensis NRRL 18488 as a new triacsin producer. In addition to targeted gene disruption to identify necessary genes for triacsin production, stable isotope feeding was performed in vivo to advance the understanding of N-hydroxytriazene biosynthesis.

biochemistry↗

bioRxiv2018

Metabolic proof-reading in Plasmodium berghei: essentiality of phosphoglycolate phosphatase

Plasmodium falciparum (Pf)4-nitrophenylphosphatase was earlier shown to be involved in vitamin B1 metabolism by Knockel et al., (Mol. Biochem. Parasitol. 2008, 157, 241-243). An independent BLASTp search showed that the protein had significant homology with phos-phoglycolate phosphatase from mouse, human and yeast, and prompted us to re-investigate the biochemical properties of the recombinant Plasmodium enzyme. Owing to the insoluble nature of the Pf enzyme, an extended substrate screen and biochemical characterization was performed on its P. berghei (Pb) homolog that led to the identification of 2-phosphoglycolate and 2-phospho L-lactate as the relevant physiological substrates. 2-phosphoglycolate is known to be generated during repair of damaged DNA ends whereas, 2-phospho L-lactate is a product of pyruvate kinase side reaction. These metabolites are potent inhibitors of the key glycolytic enzymes, triosephosphate isomerase and phosphofructokinase, and hence clearance of these toxic metabolites is vital for cell survival and functioning. Gene knockout studies conducted in P. berghei revealed the essential nature of this conserved metabolic proof-reading enzyme.

biochemistry↗

bioRxiv2018

Separate Compartments for Chromosome Entrapment and DNA Binding during SMC translocation

Multi-subunit SMC ATPase complexes translocate on chromosomal DNA. They control chromosome structure and DNA topology, presumably by acting as DNA extrusion motors. The SMC-kleisin ring entraps chromosomal DNA. The ring lumen is strongly reduced in size by alignment of the SMC arms and upon ATP binding is divided in two by engagement of SMC head domains. Here, we provide evidence for DNA binding in the SMC compartment and chromosome entrapment in the Kleisin compartment of B. subtilis Smc/ScpAB. We show that DNA binding at the Smc hinge is dispensable and identify an essential DNA binding site at engaged heads which faces the S compartment. Mutations interfering with DNA binding do not prevent ATP hydrolysis but block DNA translocation by Smc/ScpAB. Our findings are consistent with the notion that Smc/DNA contacts stabilize looped DNA segments in the S compartment, while the base of a chromosomal DNA loop is enclosed in the K compartment. Transfer of DNA double helices between S and K compartments may support DNA translocation.

biochemistry↗

bioRxiv2018

Structures of the Human Spliceosomes Before and After Release of the Ligated Exon

Pre-mRNA splicing is executed by the spliceosome, which has eight major functional states each with distinct composition. Five of these eight human spliceosomal complexes, all preceding exon ligation, have been structurally characterized. In this study, we report the cryo-electron microscopy structures of the human post-catalytic spliceosome (P complex) and intron lariat spliceosome (ILS) at average resolutions of 3.0 and 2.9 [A], respectively. In the P complex, the ligated exon remains anchored to loop I of U5 small nuclear RNA, and the 3-splice site is recognized by the junction between the 5-splice site and the branch point sequence. The ATPase/helicase Prp22, along with the ligated exon and eight other proteins, are dissociated in the P-to-ILS transition. Intriguingly, the ILS complex exists in two distinct conformations, one with the ATPase/helicase Prp43 and one without. Comparison of these three late-stage human spliceosomes reveals mechanistic insights into exon release and spliceosome disassembly.

biochemistry↗

bioRxiv2018

Optimization of accelerated solvent extraction of fatty acids from Coix seeds using chemometrics methods

This study investigated the optimization of accelerated solvent extraction (ASE) of fatty acids (FAs) from three Coix seeds (small Coix seed, SCS; big Coix seed, BCS; translucent Coix seed, TCS) by chemometrics methods. Partial least-squares regression (PLSR) and backpropagation neural network (BPNN) were applied to build models that reflect the relationship between content of FAs and extraction conditions (temperature, time, and extraction solvent). Genetic algorithms (GAs) and particle swarm optimization (PSO) were utilized to optimize the combination of extraction conditions. The composition of FAs was analysed by gas chromatography-mass spectrometry (GC-MS). The PLSR models could reflect the relationship of FA content in both BCS and SCS and extraction conditions well, while the BPNN model was more suitable for TCS. The optimal extraction conditions for BCS and SCS were obtained by GAs, whereas those of TCS were obtained by PSO. The FA compositions of the three Coix seeds exhibited differences. The results show that ASE combined with chemometrics methods can rapidly and effectively obtain the optimal conditions for the extraction of FAs from Coix seed and there are differences in the extraction conditions and compositions of FAs among different varieties of Coix seed, but all the extraction time is shorter than other extractions methods.

biochemistry↗