Large Polypeptide Research Articles

The relative energies of polypeptide conformers predicted by linear scaling second-order Møller-Plesset perturbation theory

We describe an implementation of the cluster-in-molecule (CIM) resolution of the identity (RI) approximation second-order Moller-Plesset perturbation theory (CIM-RI-MP2), with the purpose of extending RI-MP2 calculations to very large systems. For typical conformers of several large polypeptides, we calculated their conformational energy differences with the CIM-RI-MP2 and the generalized energy-based fragmentation MP2 (GEBF-MP2) methods, and compared these results with the density functional theory (DFT) results obtained with several popular functionals. Our calculations show that the conformational energy differences obtained with CIM-RI-MP2 and GEBF-MP2 are very close to each other. In comparison with the GEBF-MP2 and CIM-RI-MP2 relative energies, we found that the DFT functionals (CAM-B3LYP-D3, LC-ωPBE-D3, M05-2X, M06-2X and ωB97XD) can give quite accurate conformational energy differences for structurally similar conformers, but provide less-accurate results for structurally very different conformers.

Middle-down hybrid chromatography/tandem mass spectrometry workflow for characterization of combinatorial post-translational modifications in histones.

We present an integrated middle-down proteomics platform for sensitive mapping and quantification of coexisting PTMs in large polypeptides (5-7 kDa). We combined an RP trap column with subsequent weak cation exchange-hydrophilic interaction LC interfaced directly to high mass accuracy ESI MS/MS using electron transfer dissociation. This enabled automated and efficient separation and sequencing of hypermodified histone N-terminal tails for unambiguous localization of combinatorial PTMs. We present Histone Coder and IsoScale software to extract, filter, and analyze MS/MS data, including quantification of cofragmenting isobaric polypeptide species. We characterized histone tails derived from murine embryonic stem cells knockout in suppressor of zeste12 (Suz12(-/-) ) and quantified 256 combinatorial histone marks in histones H3, H4, and H2A. Furthermore, a total of 713 different combinatorial histone marks were identified in purified histone H3. We measured a seven-fold reduction of H3K27me2/me3 (where me2 and me3 are dimethylation and trimethylation, respectively) in Suz12(-) (/) (-) cells and detected significant changes of the relative abundance of 16 other single PTMs of histone H3 and other combinatorial marks. We conclude that the inactivation of Suz12 is associated with changes in the abundance of not only H3K27 methylation but also multiple other PTMs in histone H3 tails.

Roundabout 1 exists predominantly as a basal dimeric complex and this is unaffected by binding of the ligand Slit2

Robo (Roundabout) receptors and their Slit polypeptide ligands are known to play key roles in neuronal development and have been implicated in both angiogenesis and cancer. Like the other family members, Robo1 is a large single transmembrane domain polypeptide containing a series of well-defined extracellular elements. However, the intracellular domain lacks structural definition and little is known about the quaternary structure of Robo receptors or how binding of a Slit might affect this. To address these questions combinations of both autofluorescent protein-based FRET imaging and time-resolved FRET were employed. Both approaches identified oligomeric organization of Robo1 that did not require the presence of the intracellular domain. SpIDA (spatial intensity distribution analysis) of eGFP-tagged forms of Robo1 indicated that for a C-terminally deleted version approximately two-thirds of the receptor was present as a dimer and one-third as a monomer. By contrast, full-length Robo1 was present almost exclusively as a dimer. In each case this was unaffected by the addition of Slit2, although parallel studies demonstrated the biological activity of Slit2 and its interaction with Robo1. Deletion of both the immunoglobulin and fibronectin typeIII extracellular repeats prevented dimer formation, with the immunoglobulin repeats providing the bulk of the protein-protein interaction affinity.

Elastin-like polypeptides: The influence of its molecular weight on local hyperthermia-induced tumor accumulation

Elastin-like polypeptides (ELP) are thermally responsive polypeptides that are soluble in solutions at 37°C, but which aggregate above 42°C. ELP can be used as effective carrier systems of anticancer molecules, because they can be targeted to tumor sites through the application of local hyperthermia. Since molecular size largely influences how successfully therapeutic agents can cross the vasculatures of tumors, it was crucial to determine an optimal molecular size. In this study, we designed and evaluated three ELP macromolecules with varying molecular weights (43, 63, and 122kDa), with the goal of determining which would optimize the ELP drug delivery system. The N-terminus of the ELP macromolecule was modified with the cell penetrating peptide Bac to enhance intratumoral and intracellular uptake, and it was also confirmed that each polypeptide had the target transition temperature of 37–42°C and the results of the studies, using tumor-bearing mice, showed that the tumor accumulations increased in the case of all three peptides when local hyperthermia was applied, but that the elimination patterns from these tumors varied according to peptide size. Local hyperthermia was found to produce prolonged retention of all ELP conjugates in tumors except Bac-ELP43. In addition, the pharmacokinetic analysis showed that two larger polypeptides with 63 and 122kDa have increased AUC in comparison with the 43kDa polypeptide. These results suggest that, when combined with local hyperthermia, the larger ELP conjugates (63 and 122kDa) have advantages over the smaller Bac-ELP43 polypeptide in terms of enhanced permeability and higher retention effects.

The ribosome can prevent aggregation of partially folded protein intermediates: studies using the Escherichia coli ribosome.

BackgroundMolecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone ‘foldases’ that are distinct from chaperone’ holdases’ that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC) located in the domain V of the 23S rRNA of Escherichia coli ribosome (bDV RNA) is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains.ResultsWe demonstrate using Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII) and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein.ConclusionThe ribosome can behave like a ‘holdase’ chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.

Evidence for Proteomic and Metabolic Adaptations Associated with Alterations of Seed Yield and Quality in Sulfur-limited Brassica napus L

In Brassica napus, seed yield and quality are related to sulfate availability, but the seed metabolic changes in response to sulfate limitation remain largely unknown. To address this question, proteomics and biochemical studies were carried out on mature seeds obtained from plants grown under low sulfate applied at the bolting (LS32), early flowering (LS53), or start of pod filling (LS70) stage. The protein quality of all low-sulfate seeds was reduced and associated with a reduction of S-rich seed storage protein accumulation (as Cruciferin Cru4) and an increase of S-poor seed storage protein (as Cruciferin BnC1). This compensation allowed the protein content to be maintained in LS70 and LS53 seeds but was not sufficient to maintain the protein content in LS32 seeds. The lipid content and quality of LS53 and LS32 seeds were also affected, and these effects were primarily associated with a reduction of C18-derivative accumulation. Proteomics changes related to lipid storage, carbohydrate metabolism, and energy (reduction of caleosins, phosphoglycerate kinase, malate synthase, ATP-synthase β-subunit, and thiazole biosynthetic enzyme THI1 and accumulation of β-glucosidase and citrate synthase) provide insights into processes that may contribute to decreased oil content and altered lipid composition (in favor of long-chain fatty acids in LS53 and LS32 seeds). These data indicate that metabolic changes associated with S limitation responses affect seed storage protein composition and lipid quality. Proteins involved in plant stress response, such as dehydroascorbate reductase and Cu/Zn-superoxide dismutase, were also accumulated in LS53 and LS32 seeds, and this might be a consequence of reduced glutathione content under low S availability. LS32 treatment also resulted in (i) reduced germination vigor, as evidenced by lower germination indexes, (ii) reduced seed germination capacity, related to a lower seed viability, and (iii) a strong decrease of glyoxysomal malate synthase, which is essential for the use of fatty acids during seedling establishment.

Investigation of SecY protein‐translocation channel in action using a novel in vivo tool (362.3)

Many proteins are targeted by signal sequences to a conserved protein‐conducting channel, formed by the prokaryotic SecY or eukaryotic Sec61 complex, and are translocated across the membrane. The channel forms an hourglass‐shaped pore with a constriction in the middle across the membrane. The channel also has a lateral gate that can open toward the lipid phase. During initiation of translocation, a ribosome‐nascent chain complex binds to the SecY/Sec61 channel, resulting in insertion of the nascent chain. To study the SecY channel engaged in translocation, we developed a novel method generating defined translocation intermediate complexes in E. coli cells. Using this tool, we addressed how the channel is opened by a ribosome‐bound nascent chain and transports large polypeptides and yet prevents passage of small molecules. We determined structures of inactive and active ribosome‐channel complexes with cryo‐electron microscopy. These structures demonstrate that the nascent chain opens the channel through mostly rigid body movements of the two halves of SecY. The polypeptide inserts as a loop into the channel, with the hydrophobic region of the signal sequence intercalated into the open lateral gate. Biochemical analysis shows that the pore residues of SecY form a gasket‐like seal around the translocating polypeptide chain, preventing the permeation of small molecules. This work was supported by HHMI and NIH.

Investigation of SecY protein‐translocation channel in action using a novel in vivo tool (LB198)

Many proteins are targeted by signal sequences to a conserved protein‐conducting channel, formed by the prokaryotic SecY or eukaryotic Sec61 complex, and are translocated across the membrane. The channel forms an hourglass‐shaped pore with a constriction in the middle across the membrane. The channel also has a lateral gate that can open toward the lipid phase. During initiation of translocation, a ribosome‐nascent chain complex binds to the SecY/Sec61 channel, resulting in insertion of the nascent chain. To study the SecY channel engaged in translocation, we developed a novel method generating defined translocation intermediate complexes in E. coli cells. Using this tool, we addressed how the channel is opened by a ribosome‐bound nascent chain and transports large polypeptides and yet prevents passage of small molecules. We determined structures of inactive and active ribosome‐channel complexes with cryo‐electron microscopy. These structures demonstrate that the nascent chain opens the channel through mostly rigid body movements of the two halves of SecY. The polypeptide inserts as a loop into the channel, with the hydrophobic region of the signal sequence intercalated into the open lateral gate. Biochemical analysis shows that the pore residues of SecY form a gasket‐like seal around the translocating polypeptide chain, preventing the permeation of small molecules. This work was supported by HHMI and NIH.

Performance of density functionals for computation of core electron binding energies in first-row hydrides and glycine

A number of density functionals are benchmarked for calculation of 1s core electron binding energies for carbon, nitrogen, and oxygen nuclei in glycine, and for comparison in the first-row hydrides methane, ammonia, and water. The goal is to establish methods having potential to aid the analysis of experimental X-ray photoelectron spectra on compounds such as amino acids, DNA nucleosides, and large polypeptides in various environments. Several promising density functionals are identified that can reproduce experimental results within 0.2 eV on average for the absolute binding energies and also for the intramolecular and intermolecular shifts in the studied molecules.

Human LDL Structural Diversity Studied by IR Spectroscopy

Lipoproteins are responsible for cholesterol traffic in humans. Low density lipoprotein (LDL) delivers cholesterol from liver to peripheral tissues. A misleading delivery can lead to the formation of atherosclerotic plaques. LDL has a single protein, apoB-100, that binds to a specific receptor. It is known that the failure associated with a deficient protein-receptor binding leads to plaque formation. ApoB-100 is a large single lipid-associated polypeptide difficulting the study of its structure. IR spectroscopy is a technique suitable to follow the different conformational changes produced in apoB-100 because it is not affected by the size of the protein or the turbidity of the sample. We have analyzed LDL spectra of different individuals and shown that, even if there are not big structural changes, a different pattern in the intensity of the band located around 1617 cm−1 related with strands embedded in the lipid monolayer, can be associated with a different conformational rearrangement that could affect to a protein interacting region with the receptor.

Ubiquitin code assembly and disassembly

Ubiquitin, a 76 amino-acid polypeptide, presents a compact three-dimensional structure, utilising a fold that recurs within larger polypeptides and in other protein modifiers, such as NEDD8 and SUMO. Ubiquitylation was initially recognised as a signal for proteasome-mediated degradation. We shall consider here how this view has evolved to appreciate that the dynamic appendage of different types of ubiquitin chains represents a versatile, three-dimensional code, fundamental to the control of many cellular processes.

Isolation and molecular characterization of the shikimate dehydrogenase domain from the Toxoplasma gondii AROM complex

The apicomplexan parasite Toxoplasma gondii, the etiologic agent of toxoplasmosis, is estimated to infect 10–80% of different human populations. T. gondii encodes a large pentafunctional polypeptide known as the AROM complex which catalyzes five reactions in the shikimate pathway, a metabolic pathway required for the biosynthesis of the aromatic amino acids and a promising target for anti-parasitic agents. Here, we present the isolation, cloning and kinetic characterization of the shikimate dehydrogenase domain (TgSDH) from the T. gondii AROM complex. Recombinant TgSDH catalyzed the NADP+-dependent oxidation of shikimate in the absence of the remaining AROM domains and was sensitive to inhibition by a previously identified SDH inhibitor. Analysis of the TgSDH amino acid sequence revealed a number of novel insertions not found in SDH homologs from other organisms. Nevertheless, a three-dimensional structural model of TgSDH predicts a high level of conservation in the ‘core’ structure of the enzyme.

Getting CAD in Shape: The Atomic Structure of Human Dihydroorotase Domain

CAD is a large multifunctional polypeptide that initiates and controls the de novo biosynthesis of pyrimidines in animals. In this issue of Structure, Grande-García and colleagues provide the first atomic information of this antitumoral target by reporting the crystal structure of the dihydroorotase domain of human CAD.

Cadherin-23 May Be Dynamic in Hair Bundles of the Model Sea Anemone Nematostella vectensis

Cadherin 23 (CDH23), a component of tip links in hair cells of vertebrate animals, is essential to mechanotransduction by hair cells in the inner ear. A homolog of CDH23 occurs in hair bundles of sea anemones. Anemone hair bundles are located on the tentacles where they detect the swimming movements of nearby prey. The anemone CDH23 is predicted to be a large polypeptide featuring a short exoplasmic C-terminal domain that is unique to sea anemones. Experimentally masking this domain with antibodies or mimicking this domain with free peptide rapidly disrupts mechanotransduction and morphology of anemone hair bundles. The loss of normal morphology is accompanied, or followed by a decrease in F-actin in stereocilia of the hair bundles. These effects were observed at very low concentrations of the reagents, 0.1–10 nM, and within minutes of exposure. The results presented herein suggest that: (1) the interaction between CDH23 and molecular partners on stereocilia of hair bundles is dynamic and; (2) the interaction is crucial for normal mechanotransduction and morphology of hair bundles.

Signaling pathways involved in carcinogenesis in a double hit mouse model of cholangiopathy and hepatitis B virus

Background and aim: Primary sclerosing cholangitis (PSC) is one of the most famous liver cholestatic diseases, which leads to liver cirrhosis and liver failure. Hepatitis B virus (HBV) also leads to liver cirrhosis and hepatocellular carcinoma (HCC). Abcb4 knock out (Abcb4-/-) mice are known to develop cholangiopathy represented by portal tract inflammation and a severe ductal proliferation and developed nodules and HCC. Also, HBV transgenic mice (HBV+/-), which show liver specific expression of the large envelope polypeptide of human HBV, lead to liver damage and HCC. We already reported that these mice lead to liver inflammation and fibrosis. The aim of the present study is to clarify the signaling pathways involved in carcinogenesis in one-year old Abcb4-/-/HBV+/- hybrid mice.

Mechanisms of Dynein-Driven Microtubule Sliding and Cargo Transport

Dynein is a minus-end-directed microtubule motor protein that powers the beating of cilia and flagella and transports a wide variety of cargoes within the cytoplasm of eukaryotic cells. Dynein is not evolutionarily related to the cytoskeletal motor proteins kinesin and myosin, but instead is a member of the AAA+ superfamily. Unlike most AAA+ ATPases that self-assemble into homo-hexameric rings, dynein has six distinct AAA+ domains that are concatenated within a single, large polypeptide chain; extending from one of the AAA+ domains is a long, anti-parallel coiled-coil extension (called the stalk) that binds to microtubules. We have studied the mechanism of dynein motility using a combination of structural and single molecule studies. I will discuss how the two motor domains of dynein can act to slide apart anti-parallel microtubules, an activity that may be important for dynein's actions in organizing the mitotic spindle during mitosis. I also will discuss how dynein's enzymatic cycle might be regulated for normal cargo transport.

The crystal structure of BlmI as a model for nonribosomal peptide synthetase peptidyl carrier proteins.

Carrier proteins (CPs) play a critical role in the biosynthesis of various natural products, especially in nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymology, where the CPs are referred to as peptidyl-carrier proteins (PCPs) or acyl-carrier proteins (ACPs), respectively. CPs can either be a domain in large multifunctional polypeptides or standalone proteins, termed Type I and Type II, respectively. There have been many biochemical studies of the Type I PKS and NRPS CPs, and of Type II ACPs. However, recently a number of Type II PCPs have been found and biochemically characterized. In order to understand the possible interaction surfaces for combinatorial biosynthetic efforts we crystallized the first characterized and representative Type II PCP member, BlmI, from the bleomycin biosynthetic pathway from Streptomyces verticillus ATCC 15003. The structure is similar to CPs in general but most closely resembles PCPs. Comparisons with previously determined PCP structures in complex with catalytic domains reveals a common interaction surface. This surface is highly variable in charge and shape, which likely confers specificity for interactions. Previous nuclear magnetic resonance (NMR) analysis of a prototypical Type I PCP excised from the multimodular context revealed three conformational states. Comparison of the states with the structure of BlmI and other PCPs reveals that only one of the NMR states is found in other studies, suggesting the other two states may not be relevant. The state represented by the BlmI crystal structure can therefore serve as a model for both Type I and Type II PCPs.

Molecular and Biochemical Analyses of CbCel9A/Cel48A, a Highly Secreted Multi-Modular Cellulase by Caldicellulosiruptor bescii during Growth on Crystalline Cellulose

During growth on crystalline cellulose, the thermophilic bacterium Caldicellulosiruptor bescii secretes several cellulose-degrading enzymes. Among these enzymes is CelA (CbCel9A/Cel48A), which is reported as the most highly secreted cellulolytic enzyme in this bacterium. CbCel9A/Cel48A is a large multi-modular polypeptide, composed of an N-terminal catalytic glycoside hydrolase family 9 (GH9) module and a C-terminal GH48 catalytic module that are separated by a family 3c carbohydrate-binding module (CBM3c) and two identical CBM3bs. The wild-type CbCel9A/Cel48A and its truncational mutants were expressed in Bacillus megaterium and Escherichia coli, respectively. The wild-type polypeptide released twice the amount of glucose equivalents from Avicel than its truncational mutant that lacks the GH48 catalytic module. The truncational mutant harboring the GH9 module and the CBM3c was more thermostable than the wild-type protein, likely due to its compact structure. The main hydrolytic activity was present in the GH9 catalytic module, while the truncational mutant containing the GH48 module and the three CBMs was ineffective in degradation of either crystalline or amorphous cellulose. Interestingly, the GH9 and/or GH48 catalytic modules containing the CBM3bs form low-density particles during hydrolysis of crystalline cellulose. Moreover, TM3 (GH9/CBM3c) and TM2 (GH48 with three CBM3 modules) synergistically hydrolyze crystalline cellulose. Deletion of the CBM3bs or mutations that compromised their binding activity suggested that these CBMs are important during hydrolysis of crystalline cellulose. In agreement with this observation, seven of nine genes in a C. bescii gene cluster predicted to encode cellulose-degrading enzymes harbor CBM3bs. Based on our results, we hypothesize that C. bescii uses the GH48 module and the CBM3bs in CbCel9A/Cel48A to destabilize certain regions of crystalline cellulose for attack by the highly active GH9 module and other endoglucanases produced by this hyperthermophilic bacterium.

Celiac Disease and Metabolic Bone Disease

Celiac disease is a common autoimmune gastrointestinal disorder affecting multiple organs, precipitated in genetically vulnerable persons by the ingestion of gluten. Gluten is poorly digested and is presented to the intestinal mucosa as a large polypeptide. Binding to human leukocyte antigen-DQ2 and human leukocyte antigen-DQ8 molecules on antigen-presenting cells stimulates cellular and humeral immune reactions. Although common serological tests are available to diagnose celiac disease, the diagnosis of celiac disease is often delayed or missed because of lack of recognition as the disease presentation in adults is highly variable and may be asymptomatic. Celiac disease is a common secondary cause of metabolic bone disease and delayed treatment with gluten-free diet affects bone mineral density and fracture risk, so it is crucial to diagnose and treat celiac disease promptly. In this article, we will review recent studies of celiac disease in adults and provide practical, easily accessible information for busy clinicians.

Three-stage biochemical selection: cloning of prototype class IIS/IIC/IIG restriction endonuclease-methyltransferase TsoI from the thermophile Thermus scotoductus

BackgroundIn continuing our research into the new family of bifunctional restriction endonucleases (REases), we describe the cloning of the tsoIRM gene. Currently, the family includes six thermostable enzymes: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI, TsoI, isolated from various Thermus sp. and two thermolabile enzymes: RpaI and CchII, isolated from mesophilic bacteria Rhodopseudomonas palustris and Chlorobium chlorochromatii, respectively. The enzymes have several properties in common. They are large proteins (molecular size app. 120 kDa), coded by fused genes, with the REase and methyltransferase (MTase) in a single polypeptide, where both activities are affected by S-adenosylmethionine (SAM). They recognize similar asymmetric cognate sites and cleave at a distance of 11/9 nt from the recognition site. Thus far, we have cloned and characterised TaqII, Tth111II, TthHB27I, TspGWI and TspDTI.ResultsTsoI REase, which originate from thermophilic Thermus scotoductus RFL4 (T. scotoductus), was cloned in Escherichia coli (E. coli) using two rounds of biochemical selection of the T. scotoductus genomic library for the TsoI methylation phenotype. DNA sequencing of restriction-resistant clones revealed the common open reading frame (ORF) of 3348 bp, coding for a large polypeptide of 1116 aminoacid (aa) residues, which exhibited a high level of similarity to Tth111II (50% identity, 60% similarity). The ORF was PCR-amplified, subcloned into a pET21 derivative under the control of a T7 promoter and was subjected to the third round of biochemical selection in order to isolate error-free clones. Induction experiments resulted in synthesis of an app. 125 kDa protein, exhibiting TsoI-specific DNA cleavage. Also, the wild-type (wt) protein was purified and reaction optima were determined.ConclusionsPreviously we identified and cloned the Thermus family RM genes using a specially developed method based on partial proteolysis of thermostable REases. In the case of TsoI the classic biochemical selection method was successful, probably because of the substantially lower optimal reaction temperature of TsoI (app. 10-15°C). That allowed for sufficient MTase activity in vivo in recombinant E. coli. Interestingly, TsoI originates from bacteria with a high optimum growth temperature of 67°C, which indicates that not all bacterial enzymes match an organism’s thermophilic nature, and yet remain functional cell components. Besides basic research advances, the cloning and characterisation of the new prototype REase from the Thermus sp. family enzymes is also of practical importance in gene manipulation technology, as it extends the range of available DNA cleavage specificities.