Sendai virus N-terminal fusion peptide consists of two similar repeats, both of which contribute to membrane fusion.

The N-terminal fusion peptide of Sendai virus F1 envelope glycoprotein is a stretch of 14 amino acids, most of which are hydrophobic. Following this region, we detected a segment of 11 residues that are strikingly similar to the N-terminal fusion peptide. We found that, when anchored to the membrane by palmitoylation of its N-terminus, this segment (WT-palm-19-33) induces membrane fusion of large unilamellar liposomes to almost the same extent as a segment that includes the N-terminal fusion peptide. The activity of WT-palm-19-33 was dependent on its specific sequence, as a palmitoylated peptide with the same amino-acid composition but a scrambled sequence was inactive. Interestingly, two mutations (G7A and G12A) known to increase F1- induced cell-cell fusion, also increased the homology between the N-terminal fusion peptide and WT-palm-19-33. The role of the amino-acid sequence on the fusogenicity, secondary structure, and mechanism of membrane fusion was analyzed by comparing a peptide comprising both homologous segments (WT 1-33), a G12A mutant (G12A 1-33), a G7A-G12A double mutant (G7A-G12A 1-33), and a peptide with a scrambled sequence (SC 1-33). Based on these experiments, we postulate that replacement of Gly 7 and Gly12 by Ala increases the alpha helical content of the N-terminal region, with a concomitant increase in its fusogenic activity. Furthermore, the dissimilar abilities of the different peptides to induce membrane negative curvature as well as to promote isotropic 31P NMR signals, suggest that these mutations might also alter the extent of membrane penetration of the 33-residue peptide. Interestingly, our results serve to explain the effect of the G7A and G12A mutations on the fusogenic activity of the parent F1 protein in vivo.     

Functional analysis of the putative fusion domain of the baculovirus envelope fusion protein F

Group II nucleopolyhedroviruses (NPVs), e.g., Spodoptera exigua MNPV, lack a GP64-like protein that is present in group I NPVs but have an unrelated envelope fusion protein named F. In contrast to GP64, the F protein has to be activated by a posttranslational cleavage mechanism to become fusogenic. In several vertebrate viral fusion proteins, the cleavage activation generates a new N terminus which forms the so-called fusion peptide. This fusion peptide inserts in the cellular membrane, thereby facilitating apposition of the viral and cellular membrane upon sequential conformational changes of the fusion protein. A similar peptide has been identified in NPV F proteins at the N terminus of the large membrane-anchored subunit F(1). The role of individual amino acids in this putative fusion peptide on viral infectivity and propagation was studied by mutagenesis. Mutant F proteins with single amino acid changes as well as an F protein with a deleted putative fusion peptide were introduced in gp64-null Autographa californica MNPV budded viruses (BVs). None of the mutations analyzed had an major effect on the processing and incorporation of F proteins in the envelope of BVs. Only two mutants, one with a substitution for a hydrophobic residue (F152R) and one with a deleted putative fusion peptide, were completely unable to rescue the gp64-null mutant. Several nonconservative substitutions for other hydrophobic residues and the conserved lysine residue had only an effect on viral infectivity. In contrast to what was expected from vertebrate virus fusion peptides, alanine substitutions for glycines did not show any effect.     

Effect of nonpolar substitutions of the conserved Phe11 in the fusion peptide of HIV-1 gp41 on its function, structure, and organization in membranes.

The fusion domain of the HIV-1 envelope glycoprotein (gp120-gp41) is a conserved hydrophobic region located at the N-terminus of the transmembrane subunit (gp41). A prominent feature of this domain is a conserved five-residue "FLGFL" sequence at positions 8-12. Mutation of the highly conserved Phe(11) to Val (F11V), presumed not to significantly affect the hydrophobicity and the structure of this region, has been shown to decrease the level of syncytium formation and virus infectivity. Here we show that the substitution of Gly for Phe(11) (F11G) reduces cell-cell fusion activity by 80-90%. To determine the effect of these mutations on the properties of the fusion peptide, a 33-residue peptide (WT) identical to the extended fusion domain and its F11V and F11G mutants were synthesized, fluorescently labeled, and studied with respect to their function, structure, and organization in phospholipid membranes. The WT peptide alone induced fusion of both zwitterionic (PC/Chol) and negatively charged (PS/PC/Chol and POPG) vesicles, in contrast to a 23-mer fusion peptide lacking the C-terminal domain which has been shown to be inactive with PC vesicles but able to induce fusion of POPG vesicles which had been preaggragated with Ca(2+) or Mg(2+). The F11V peptide preserved 50% activity, and the F11G peptide was virtually inactive. ATR-FTIR spectroscopy indicated similar secondary structure of the peptides in multibilayers that was independent of membrane composition. Furthermore, all the peptides increased the extent of lipid disorder to a similar extent, but the kinetics of amide II H to D exchange was in the following order: F11G > F11V > WT. Fluorescence studies in the presence of membranes, as well as SDS-PAGE, revealed that the WT and F11V peptides self-associate to similar levels while F11G exhibited a decreased level of self-association. The data suggest that the FLGFL motif contributes to the functional organization of the HIV-1 fusion peptide and that the C-terminal domain following the fusion peptide contributes to the membrane fusion process.     

Determination of the membrane contact residues and solution structure of the helix F/G loop of prostaglandin I2 synthase

From our topological arrangement model of prostaglandin I(2) synthase (PGIS) created by homology modeling and topology studies, we hypothesized that the helix F/G loop of PGIS contains a membrane contact region distinct from the N-terminal membrane anchor domain. To provide direct experimental data we have explored the relationship between the endoplasmic reticulum (ER) membrane and the PGIS F/G loop using a constrained synthetic peptide to mimic PGIS residues 208-230 cyclized on both ends through a disulfide bond with added Cys residues. The solution structure and the residues important for membrane contact of the constrained PGIS F/G loop peptide were investigated by high-resolution 1H two-dimensional nuclear magnetic resonance (2D NMR) experiments and a spin label incorporation technique. Through the combination of 2D NMR experiments in the presence of dodecylphosphocholine (DPC) micelles used to mimic the membrane environment, complete 1H NMR assignments of the F/G loop segment have been obtained and the solution structure of the peptide has been determined. The PGIS F/G loop segment shows a defined helix turn helix conformation, which is similar to the three-dimensional crystallography structure of P450BM3 in the corresponding region. The orientation and the residues contacted with the membrane of the PGIS F/G loop were evaluated from the effect of incorporation of a spin-labeled 12-doxylstearate into the DPC micelles with the peptide. Three residues in the peptide corresponding to the PGIS residues L217 (L11), L222 (L16), and V224 (V18) have been demonstrated to contact the DPC micelles, which implies that the residues are involved in contact with the ER membrane in the native membrane-bound PGIS. These results provided the first experimental evidence to localize the membrane contact residues in the F/G loop region of microsomal P450 and are valuable to further define and understand the membrane topology of PGIS and those of other microsomal P450s in the native membrane environment.     

Spring-loaded heptad repeat residues regulate the expression and activation of paramyxovirus fusion protein

During viral entry, the paramyxovirus fusion (F) protein fuses the viral envelope to a cellular membrane. Similar to other class I viral fusion glycoproteins, the F protein has two heptad repeat regions (HRA and HRB) that are important in membrane fusion and can be targeted by antiviral inhibitors. Upon activation of the F protein, HRA refolds from a spring-loaded, crumpled structure into a coiled coil that inserts a hydrophobic fusion peptide into the target membrane and binds to the HRB helices to form a fusogenic hairpin. To investigate how F protein conformational changes are regulated, we mutated in the Sendai virus F protein a highly conserved 10-residue sequence in HRA that undergoes major structural changes during protein refolding. Nine of the 15 mutations studied caused significant defects in F protein expression, processing, and fusogenicity. Conversely, the remaining six mutations enhanced the fusogenicity of the F protein, most likely by helping spring the HRA coil. Two of the residues that were neither located at "a" or "d" positions in the heptad repeat nor conserved among the paramyxoviruses were key regulators of the folding and fusion activity of the F protein, showing that residues not expected to be important in coiled-coil formation may play important roles in regulating membrane fusion. Overall, the data support the hypothesis that regions in the F protein that undergo dramatic changes in secondary and tertiary structure between the prefusion and hairpin conformations regulate F protein expression and activation.     

15N NMR study of the ionization properties of the influenza virus fusion peptide in zwitterionic phospholipid dispersions

Influenza virus hemagglutinin (HA)-mediated membrane fusion involves insertion into target membranes of a stretch of amino acids located at the N-terminus of the HA(2) subunit of HA at low pH. The pK(a) of the alpha-amino group of (1)Gly of the fusion peptide was measured using (15)N NMR. The pK(a) of this group was found to be 8.69 in the presence of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The high value of this pK(a) is indicative of stabilization of the protonated form of the amine group through noncovalent interactions. The shift reagent Pr(3+) had large effects on the (15)N resonance from the alpha-amino group of Gly(1) of the fusion peptide in DOPC vesicles, indicating that the terminal amino group was exposed to the bulk solvent, even at low pH. Furthermore, electron paramagnetic resonance studies on the fusion peptide region of spin-labeled derivatives of a larger HA construct are consistent with the N-terminus of this peptide being at the depth of the phosphate headgroups. We conclude that at both neutral and acidic pH, the N-terminal of the fusion peptide is close to the aqueous phase and is protonated. Thus neither a change in the state of ionization nor a significant increase in membrane insertion of this group is associated with increased fusogenicity at low pH.     

Conserved leucines in N-terminal heptad repeat HR1 of envelope fusion protein F of group II nucleopolyhedroviruses are important for correct processing and essential for fusogenicity

The heptad repeat (HR), a conserved structural motif of class I viral fusion proteins, is responsible for the formation of a six-helix bundle structure during the envelope fusion process. The insect baculovirus F protein is a newly found budded virus envelope fusion protein which possesses common features to class I fusion proteins, such as proteolytic cleavage and the presence of an N-terminal open fusion peptide and multiple HR domains on the transmembrane subunit F(1). Similar to many vertebrate viral fusion proteins, a conserved leucine zipper motif is predicted in this HR region proximal to the fusion peptide in baculovirus F proteins. To facilitate our understanding of the functional role of this leucine zipper-like HR1 domain in baculovirus F protein synthesis, processing, and viral infectivity, key leucine residues (Leu209, Leu216, and Leu223) were replaced by alanine (A) or arginine (R), respectively. By using Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) as a pseudotype expression system, we demonstrated that all mutant F proteins incorporated into budded virus, indicating that leucine substitutions did not affect intercellular trafficking of F. Furin-like protease cleavage was not affected by any of the leucine substitutions; however, the disulfide bridging and N-linked glycosylation patterns were partly altered. Single substitutions in HR1 showed that the three leucine residues were critical for F fusogenicity and the rescue of AcMNPV infectivity. Our results support the view that the leucine zipper-like HR1 domain is important to safeguard the proper folding, glycosylation, and fusogenicity of baculovirus F proteins.     

1H and 15N NMR characterization of free and bound states of an amphiphilic peptide interacting with calmodulin.

A peptide of 17 amino acid residues Ac-L-K-W-K-K-L-L-K-L-L-K-K-L-L-K-L-G-NH2, designed to form an amphiphilic basic alpha-helix [DeGrado, W.F., Prendergast, F. G., Wolfe, H. R., Jr., & Cox, J. A. (1985) J. Cell. Biochem. 29, 83-93], was labeled with 15N at positions 1, 7, 9, and 10. Homo- and heteronuclear NMR techniques were used to characterize the conformational changes of the peptide when it binds to calmodulin in the presence of Ca2+ ions. The spectrum of the free peptide in aqueous solution at pH 6.3 and 298 K was completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide populates, to a significant extent, an alpha-helix conformational state, in agreement with circular dichroism measurements under similar physicochemical conditions. 15N-edited 1D spectra and 15N(omega 2)-half-filtered two-dimensional NMR experiments on the peptide in a 1:1 complex with calmodulin allowed assignment of half of the amide proton resonances and three C alpha H resonances of the bound peptide. The observed NOE connectivities between the peptide backbone protons are indicative of a stable helical secondary structure spanning at least the fragment L1-K11. The equilibrium and dynamic NMR parameters of the bound peptide are discussed in terms of a molecular interaction model.     

A peptide from the eel pancreas with structural similarity to human pancreatic secretory trypsin inhibitor

The primary structure of a 61-amino-acid residue peptide from the pancreas of the European eel (Anguilla anguilla) has been established as E E K S G(5)L Y R K P(10)S C G E M(15)S A M H A(20)C P M N F(25)A P V C G(30)T D G N T(35)Y P N E C(40)S L C F Q(45)R Q N T K(50)T D I L I(55)T K D D R(60)C. There was no indication of microheterogeneity. This peptide shows structural similarity to pancreatic secretory trypsin inhibitors from several mammalian species and to a cholecystokinin-releasing peptide isolated from rat pancreatic juice. A comparison of the amino acid sequences of the peptides has identified a domain in the central region of the molecules that has been strongly conserved during evolution. In contrast, the amino acid sequence in the region corresponding to the reactive centre of the mammalian trypsin inhibitors is very poorly conserved in the eel peptide. The P1-P1' reactive site lysine-isoleucine (or arginine-isoleucine) bond in the mammalian trypsin inhibitors is replaced by a methionine-asparagine bond. This region does, however, show limited homology to the reactive centre of human alpha 1-protease inhibitor suggesting that the eel peptide may function as an inhibitor of other proteolytic enzymes in the pancreas.     

Processing of HEBP1 by cathepsin D gives rise to F2L, the agonist of formyl peptide receptor 3

The peptide F2L was previously characterized as a high-affinity natural agonist for the human formyl peptide receptor (FPR) 3. F2L is an acetylated 21-aa peptide corresponding with the N terminus of the intracellular heme-binding protein 1 (HEBP1). In the current work, we have investigated which proteases were able to generate the F2L peptide from its precursor HEBP1. Structure-function analysis of F2L identified three amino acids, G(3), N(7), and S(8), as the most important for interaction of the peptide with FPR3. We expressed a C-terminally His-tagged form of human HEBP1 in yeast and purified it to homogeneity. The purified protein was used as substrate to identify proteases generating bioactive peptides for FPR3-expressing cells. A conditioned medium from human monocyte-derived macrophages was able to generate bioactivity from HEBP1, and this activity was inhibited by pepstatin A. Cathepsin D was characterized as the protease responsible for HEBP1 processing, and the bioactive product was identified as F2L. We have therefore determined how F2L, the specific agonist of FPR3, is generated from the intracellular protein HEBP1, although it is unknown in which compartment the processing by cathepsin D occurs in vivo.     

Solid-state nuclear magnetic resonance evidence for an extended beta strand conformation of the membrane-bound HIV-1 fusion peptide.

Solid-state nuclear magnetic resonance (NMR) spectroscopy was applied to the membrane-bound form of a synthetic peptide representing the 23-residue N-terminal fusion peptide domain of the HIV-1 gp41 envelope glycoprotein. 1D solid-state NMR line width measurements of singly 13C carbonyl labeled peptides showed that a significant population of the membrane-bound peptide is well-structured in its N-terminal and central regions while the C-terminus has more disorder. There was some dependence of line width on lipid composition, with narrower line widths and hence greater structural order observed for a lipid composition comparable to that found in the virus and its target T cells. In the more ordered N-terminal and central regions of the peptide, the 13C carbonyl chemical shifts are consistent with a nonhelical membrane-bound conformation. Additional evidence for a beta strand membrane-bound conformation was provided by analysis of 2D rotor-synchronized magic angle spinning NMR spectra of doubly 13C carbonyl labeled peptides. Lipid mixing and aqueous contents leakage assays were applied to demonstrate the fusogenicity of the peptide under conditions comparable to those used for the solid-state NMR sample preparation.     

A solid-state NMR study of changes in lipid phase induced by membrane-fusogenic LV-peptides

Membrane fusion requires restructuring of lipid bilayers mediated by fusogenic membrane proteins. Peptides that correspond to natural transmembrane sequences or that have been designed to mimic them, such as low-complexity “Leu-Val” (LV) peptide sequences, can drive membrane fusion, presumably by disturbing the lipid bilayer structure. Here, we assess how peptides of different fusogenicity affect membrane structure using solid state NMR techniques. We find that the more fusogenic variants induce an unaligned lipid phase component and a large degree of phase separation as observed in ³¹P 2D spectra. The data support the idea that fusogenic peptides accumulate PE in a non-bilayer phase which may be critical for the induction of fusion.     

Mutational Analysis of the Putative Fusion Domain of Ebola Virus Glycoprotein

Ebola viruses contain a single glycoprotein (GP) spike, which functions as a receptor binding and membrane fusion protein. It contains a highly conserved hydrophobic region (amino acids 524 to 539) located 24 amino acids downstream of the N terminus of the Ebola virus GP2 subunit. Comparison of this region with the structural features of the transmembrane subunit of avian retroviral GPs suggests that the conserved Ebola virus hydrophobic region may, in fact, serve as the fusion peptide. To test this hypothesis directly, we introduced conservative (alanine) and nonconservative (arginine) amino acid substitutions at eight positions in this region of the GP2 molecule. The effects of these mutations were deduced from the ability of the Ebola virus GP to complement the infectivity of a vesicular stomatitis virus (VSV) lacking the receptor-binding G protein. Some mutations, such as Ile-to-Arg substitutions at positions 532 (I532R), F535R, G536A, and P537R, almost completely abolished the ability of the GP to support VSV infectivity without affecting the transport of GP to the cell surface and its incorporation into virions or the production of virus particles. Other mutations, such as G528R, L529A, L529R, I532A, and F535A, reduced the infectivity of the VSV-Ebola virus pseudotypes by at least one-half. These findings, together with previous reports of liposome association with a peptide corresponding to positions 524 to 539 in the GP molecule, offer compelling support for a fusion peptide role for the conserved hydrophobic region in the Ebola virus GP.     

Synthesis,enhanced fusogenicity,and solid state NMR measurements of cross-linked HIV-1 fusion peptides

In the HIV-1 gp41 and other viral fusion proteins, the minimal oligomerization state is believed to be trimeric with three N-terminal fusion peptides inserting into the membrane in close proximity. Previous studies have demonstrated that the fusion peptide by itself serves as a useful model fusion system, at least to the hemifusion stage in which the viral and target cell lipids are mixed. In the present study, HIV-1 fusion peptides were chemically synthesized and cross-linked at their C-termini to form dimers or trimers. C-terminal trimerization is their likely topology in the fusogenic form of the intact gp41 protein. The fusogenicity of the peptides was then measured in an intervesicle lipid mixing assay, and the assay results were compared to those of the monomer. For monomer, dimer, and trimer at peptide strand/lipid mol ratios between 0.0050 and 0.010, the final extent of lipid mixing for the dimer and trimer was 2-3 times greater than for the monomer. These data suggest that the higher local concentration of peptide strands in the cross-linked peptides enhances fusogenicity and that oligomerization of the fusion peptide in gp41 may enhance the rate of viral/target cell membrane fusion. For gp41, this effect is in addition to the role of the trimeric coiled-coil structure in bringing about apposition of viral and target cell membranes. NMR measurements on the membrane-associated dimeric fusion peptide were consistent with an extended structure at Phe-8, which is the same as has been observed for the membrane-bound monomer in the same lipid composition.     

Coordinate deletion of N-glycans from the heptad repeats of the fusion F protein of Newcastle disease virus yields a hyperfusogenic virus with increased replication, virulence, and immunogenicity

The role of N-linked glycosylation of the Newcastle disease virus (NDV) fusion (F) protein in viral replication and pathogenesis was examined by eliminating potential acceptor sites using a reverse genetics system for the moderately pathogenic strain Beaudette C (BC). The NDV-BC F protein contains six potential acceptor sites for N-linked glycosylation at residues 85, 191, 366, 447, 471, and 541 (sites Ng1 to Ng6, respectively). The sites at Ng2 and Ng5 are present in heptad repeat (HR) domains HR1 and HR2, respectively, and thus might affect fusion. Each N-glycosylation site was eliminated individually by replacing asparagine (N) with glutamine (Q), and a double mutant (Ng2 + 5) involving the two HR domains was also made. Each mutant was successfully recovered by reverse genetics except for the one involving Ng6, which is present in the cytoplasmic domain. All of the F proteins expressed by the recovered mutant viruses were efficiently cleaved and transported to the infected-cell surface. None of the individual mutations affected viral fusogenicity, but the double mutation at Ng2 and Ng5 in HR1 and HR2 increased fusogenicity >12-fold. The single mutations at sites Ng1, Ng2, and Ng5 resulted in modestly reduced multicycle growth in vitro. These three single mutations were also the most attenuating in eggs and 1-day-old chicks and were associated with decreased replication and spread in 2-week-old chickens. In contrast, the combination of the mutations at Ng2 and Ng5 yielded a virus that, compared to the BC parent, replicated >100-fold more efficiently in vitro, was more virulent in eggs and chicks, replicated more efficiently in chickens with enhanced tropism for the brain and gut, and elicited stronger humoral cell responses. These results illustrate the effects of N-glycosylation of the F protein on NDV pathobiology and suggest that the N-glycans in HR1 and HR2 coordinately downregulate viral fusion and virulence.     

Oligomeric beta-structure of the membrane-bound HIV-1 fusion peptide formed from soluble monomers

The human immunodeficiency virus type 1 (HIV-1) fusion peptide serves as a useful model system for understanding viral/target cell fusion, at least to the lipid mixing stage. Previous solid-state NMR studies have shown that the peptide adopts an oligomeric beta-strand structure when associated with a lipid and cholesterol mixture close to that of membranes of host cells of the virus. In this study, this structure was further investigated using four different peptide constructs. In aqueous buffer solution, two of the constructs were primarily monomeric whereas the other two constructs had significant populations of oligomers/aggregates. NMR measurements for all membrane-associated peptide constructs were consistent with oligomeric beta-strand structure. Thus, constructs that are monomeric in solution can be converted to oligomers as a result of membrane association. In addition, samples prepared by very different methods had very similar NMR spectra, which indicates that the beta-strand structure is an equilibrium rather than a kinetically trapped structure. Lipid mixing assays were performed to assess the fusogenicities of the different constructs, and there was not a linear correlation between the solution oligomeric state and fusogenicity. However, the functional assays do suggest that small oligomers may be more fusogenic than either monomers or large aggregates.     

Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: Patterns revealed by three-dimensional molecular modelling of mutations of the protease domain

Familial deficiency of protein C is associated with inherited thrombophilia. To explore how specific missense mutations might cause observed clinical phenotypes, known protein C missense mutations were mapped onto three-dimensional homology models of the protein C protease domain, and the implications for domain folding and structure were evaluated. Most Type I missense mutations either replaced internal hydrophobic residues (I201T, L223F, A259V, A267T, A346T, A346V, G376D) or nearby interacting residues (I403M, T298M, Q184H), thus disrupting the packing of internal hydrophobic side chains, or changed hydrophilic residues, thus disrupting ion pairs (N256D, R178W). Mutations (P168L, R169W) at the activation site destabilized the region containing the activation peptide structure. Most Type II mutations involved solvent-exposed residues and were clustered either in a positively charged region (R147W, R157Q, R229Q, R352W) or were located in or near the active site region (S252N, D359N, G381S, G391S, H211Q). The cluster of arginines 147, 157, 229, and 352 may identify a functionally important exosite. Identification of the spatial relationships of natural mutations in the protein C model is helpful for understanding manifestations of protein C deficiency and for identification of novel, functionally important molecular features and exosites. © 1994 John Wiley & Sons, Inc.     

In vivo and in vitro characterization of TEV protease mutants

Tobacco etch virus protease (TEVp) is frequently applied in the cleavage of fusion protein. However, production of TEV protease in Escherichia coli is hampered by low yield and poor solubility, and auto-cleavage of wild type TEVp gives rise to the loss-of-function. Previously it was reported that TEVp S219V displayed more stability, and TEVp variant containing T17S/N68D/I77V and double mutant L56V/S135G resulted in the enhanced production and solubility, respectively. Here, we introduced T17S/N68D/I77V in TEVp S219V to generate TEVpM1 and combined five amino acid mutations (T17S/L56V/N68D/I77V/S135G) in TEVp S219V to create TEVpM2. Among TEVp S219V, and two constructed variants, TEVpM2 displayed highest solubility and catalytic activity in vivo, using EmGFP as the solubility reporter, and the designed fusion protein as in vivo substrate containing an N-terminal hexahistidine tagged GST, a peptide sequence for thrombin and TEV cut and E. coli diaminopropionate ammonia-lyase. The purified TEVp mutants fused with double hexahistidine-tag at N and C terminus showed highest yield, solubility and cleavage efficiency. Mutations of five amino acid residues in TEVpM2 slightly altered protein secondary structure conformed by circular dichroism assay.     

The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

Systematic Identification of H274Y Compensatory Mutations in Influenza A Virus Neuraminidase by High-Throughput Screening

Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensatory effect of E214D is applicable in both seasonal influenza virus strain A/New Caledonia/20/1999 and 2009 pandemic swine influenza virus strain A/California/04/2009. The technique described here has the potential to profile a gene at the single-nucleotide level to comprehend the dynamics of mutation space and fitness and thus offers prediction power for emerging mutant species.