Differential impact of MexB mutations on substrate selectivity of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa

The integral inner membrane resistance-nodulation-division (RND) components of three-component RND-membrane fusion protein-outer membrane factor multidrug efflux systems define the substrate selectivity of these efflux systems. To gain a better understanding of what regions of these proteins are important for substrate recognition, a plasmid-borne mexB gene encoding the RND component of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa was mutagenized in vitro by using hydroxylamine and mutations compromising the MexB contribution to antibiotic resistance identified in a DeltamexB strain. Of 100 mutants that expressed wild-type levels of MexB and showed increased susceptibility to one or more of carbenicillin, chloramphenicol, nalidixic acid, and novobiocin, the mexB genes of a representative 46 were sequenced, and 19 unique single mutations were identified. While the majority of mutations occurred within the large periplasmic loops between transmembrane segment 1 (TMS-1) and TMS-2 and between TMS-7 and TMS-8 of MexB, mutations were seen in the TMSs and in other periplasmic as well as cytoplasmic loops. By threading the MexB amino acid sequence through the crystal structure of the homologous RND transporter from Escherichia coli, AcrB, a three-dimensional model of a MexB trimer was obtained and the mutations were mapped to it. Unexpectedly, most mutations mapped to regions of MexB predicted to be involved in trimerization or interaction with MexA rather than to regions expected to contribute to substrate recognition. Intragenic second-site suppressor mutations that restored the activity of the G220S mutant version of MexB, which was compromised for resistance to all tested MexAB-OprM antimicrobial substrates, were recovered and mapped to the apparently distal portion of MexB that is implicated in OprM interaction. As the G220S mutation likely impacted trimerization, it appears that either proper assembly of the MexB trimer is necessary for OprM interaction or OprM association with an unstable MexB trimer might stabilize it, thereby restoring activity.     

Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa

The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on Pseudomonas aeruginosa. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408-->Glu/Lys939-->Arg mutant retained significant activity, while an Asp407-->Glu/Lys939-->Arg mutant exhibited only marginal function. An Asp407-->Glu/Asp408-->Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407-->Glu/Asp408-->Glu/Lys939-->Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling.     

High-resolution NMR studies of fibrinogen-like peptides in solution: structural basis for the bleeding disorder caused by a single mutation of Gly(12) to Val(12) in the A alpha chain of human fibrinogen Rouen

In human fibrinogen Rouen, which is the origin of a bleedin disorder, a single amino acid is mutated from Gly(12) to Val(12) in the A alpha chain. In the previous paper of this series, this mutation was predicted to disrupt the structure of fibrinogen-like peptides bound to bovine thrombin. The structural basis of this bleeding disorder has been further assessed by studying the interaction of the following Val(12)-substituted human fibrinogen-like peptides with bovine thrombin in aqueous solution by use of two-dimensional NMR spectroscopy (including TRNOE): Ala-Asp-Ser-Gly-Glu-Gly-Asp(7)-Phe-Leu-Ala- Glu-Val(12)-Gly-Gly-Val-Arg(16)-Gly(17)-Pro-Arg-Val-NH2 (F16), Ala-Asp-Ser-Gly-Glu-Gly-Asp(7)-Phe-Leu-Ala-Glu-Val(12)-Gly-Gly-Val- Arg(16) (tF16), Ala-Asp-Ser-Gly-Glu-Cys(Acm)-Asp(7)-Phe-Leu-Ala-Glu-Val(12)-Gly-Gly-Val- Arg(16)-Gly(17)-Pro-Arg-Val-Cys(Acm)-NH2 (F17), and Ala-Asp-Ser-Gly-Glu-Cys(Acm)-Asp(7)-Phe-Leu-Ala-Glu-Val(12)-Gly-Gly- Val-Arg(16) (tF17). Binding of thrombin to peptides F16 and F17, and hence to tF16 and tF17 as a result of the cleavage of the Arg(16)-Gly(17) peptide bond, broadens the proton resonances of residues Asp(7) to Arg(16), suggesting that thrombin interacts specifically with this sequence of residues. Medium- and long-range TRNOE's were observed between the NH proton of Asp(7) and the C beta H protons of Ala(10) and between the ring protons of Phe(8) and the C gamma H protons of Val(12) and Val(15) in complexes of thrombin with both tF16 and tF17. A strong TRNOE, in peptides tF16 and tF17, between the C beta H protons of Glu(11) and the backbone NH proton of Val(12) was also observed. However, TRNOE's between the ring protons of Phe(8) and the C alpha H protons of Gly(14) and between the C alpha H proton of Glu(11) and the NH proton of Gly(13), previously observed in the complex of thrombin with FpA, were absent in both peptides tF16 and tF17. From incorporation of TRNOE information into distance geometry calculations, Val(12) was found to disrupt the type II beta-turn involving Glu(11) and Gly(12) that is present in complexes of thrombin with normal fibrinogen-like peptides. The positions of Gly(13) and Gly(14) in the complex are also displaced, relative to the aromatic ring of Phe(8), by the Val(12) substitution. This altered geometry presumably affects the positioning of the Arg(16)-Gly(17) bond in the active site of thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Single mutation at the intersubunit interface confers extra efficiency to Cu,Zn superoxide dismutase

The Val28-->Gly single mutant at the subunit interface of Cu,Zn superoxide dismutase from Photobacterium leiognathi displays a k(cat)/K(M) value of 1.7x10(10) M(-1) s(-1), twice that of the native enzyme. Analysis of the three-dimensional structure indicates that the active site Cu,Zn center is not perturbed, slight structural deviations being only localized in proximity of the mutation site. The enzyme-substrate association rate, calculated by Brownian dynamics simulation, is identical for both enzymes, indicating that the higher catalytic efficiency of the Val28-->Gly mutant is not due to a more favorable electrostatic potential distribution. This result demonstrates the occurrence of an intramolecular communication between the mutation site and the catalytic center, about 18 A away and indicates a new strategy to encode extra efficiency within other members of this enzymatic family.     

Theoretical study about the variability of the genome of foot-and-mouth disease viruses.

1. The possibilities of change in amino acids of a protein are discussed in terms of a point mutation. 2. Whereas Met and Trp are forced to change due to a point mutation, other amino acids (Ala, Arg, Gly, Leu, Pro and Val) have a probability of 1/3 to survive in the sequence. 3. On basis of these considerations, the genome from 5 strains (CSP, C3Ind, O1K, A10 and A12) of the foot-and-mouth disease virus was studied. 4. A hypothetical genealogic tree for these strains is suggested, where CSP and C3Ind are close and also A10 and A12. O1K is closer to A10 and A12 than to CSP or C3Ind.     

Conformational effects of amino acid substitutions at positions 10, 12, and 13 in the P21 protein

Substitutions of amino acids for Gly 12 or Gly 13 in theras oncogene-encoded P21 proteins have been demonstrated to produce unique structural changes in these proteins that correlate with their ability to produce cell transformation. For example, the P21 proteins with Arg 12 or Val 13 are both known to be actively transforming. Recent site-specific mutagenesis experiments on the transforming Arg 12 protein have found that the substitution of Val for Gly 10 has no effect on transforming activity whereas the substitution of Val for Gly 13 led to a loss of transforming activity. In this study, we examine the structural effects of these substitutions on the amino terminal hydrophobic decapeptide (Leu 6-Gly 15) of P21 using conformational energy analysis. The results show that the transforming proteins with Gly 10 and Arg 12 or Val 10 and Arg 12 can both adopt the putative malignancy-causing conformation, whereas, for the nontransforming protein with Arg 12 and Val 13, this conformation is energetically disallowed. These results further support the theory that due to structural changes the transforming P21 proteins are unable to bind to some regulatory cellular element which may be the recently identified binding protein responsible for the induction of increased GTPase activity in normal P21 compared with transforming mutants.     

Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexA,B-OprM extrusion pump in Pseudomonas aeruginosa

The MexA,B-OprM efflux pump assembly of Pseudomonas aeruginosa consists of two inner membrane proteins and one outer membrane protein. The cytoplasmic membrane protein, MexB, appears to function as the xenobiotic-exporting subunit, whereas the MexA and OprM proteins are supposed to function as the membrane fusion protein and the outer membrane channel protein, respectively. Computer-aided hydropathy analyses of MexB predicted the presence of up to 17 potential transmembrane segments. To verify the prediction, we analyzed the membrane topology of MexB using the alkaline phosphatase gene fusion method. We obtained the following unique characteristics. MexB bears 12 membrane spanning segments leaving both the amino and carboxyl termini in the cytoplasmic side of the inner membrane. Both the first and fourth periplasmic loops had very long hydrophilic domains containing 311 and 314 amino acid residues, respectively. This fact suggests that these loops may interact with other pump subunits, such as the membrane fusion protein MexA and the outer membrane protein OprM. Alignment of the amino- and the carboxyl-terminal halves of MexB showed a 30% homology and transmembrane segments 1, 2, 3, 4, 5, and 6 could be overlaid with the segments 7, 8, 9, 10, 11, and 12, respectively. This result suggested that the MexB has a 2-fold repeat that strengthen the experimentally determined topology model. This paper reports the structure of the pump subunit, MexB, of the MexA,B-OprM efflux pump assembly. This is the first time to verify the topology of the resistant-nodulation-division efflux pump protein.     

Second-site suppression of a nonfunctional mutation within the Leishmania donovani inosine-guanosine transporter

LdNT2 is a member of the equilibrative nucleoside transporter family, which possesses several conserved residues located mainly within transmembrane domains. One of these residues, Asp(389) within LdNT2, was shown previously to be critical for transporter function without affecting ligand affinity or plasma membrane targeting. To further delineate the role of Asp(389) in LdNT2 function, second-site suppressors of the ldnt2-D389N null mutation were selected in yeast deficient in purine nucleoside transport and incapable of purine biosynthesis. A library of random mutants within the ldnt2-D389N background was screened in yeast for restoration of growth on inosine. Twelve different clones were obtained, each containing secondary mutations enabling inosine transport. One mutation, N175I, occurred in four clones and conferred augmented inosine transport capability compared with LdNT2 in yeast. N175I was subsequently introduced into an ldnt2-D389N construct tagged with green fluorescent protein and transfected into a Deltaldnt1/Deltaldnt2 Leishmania donovani knockout. GFP-N175I/D389N significantly suppressed the D389N phenotype and targeted properly to the plasma membrane and flagellum. Most interestingly, N175I increased the inosine K(m) by 10-fold within the D389N background relative to wild type GFP-LdNT2. Additional substitutions introduced at Asn(175) established that only large, nonpolar amino acids suppressed the D389N phenotype, indicating that suppression by Asn(175) has a specific size and charge requirement. Because multiple suppressor mutations alleviate the constraint imparted by the D389N mutation, these data suggest that Asp(389) is a conformationally sensitive residue. To impart spatial information to the clustering of second-site mutations, a three-dimensional model was constructed based upon members of the major facilitator superfamily using threading analysis. The model indicates that Asn(175) and Asp(389) lie in close proximity and that the second-site suppressor mutations cluster to one region of the transporter.     

Yeast cell death caused by mutation of the OST2 gene encoding the epsilon-subunit of Saccharomyces cerevisiae oligosaccharyltransferase

An essential epsilon-subunit of oligosaccharyltransferase Ost2 is a yeast homolog of mammalian highly conserved DAD1 (defender against apoptotic death). In hamster cells, the Gly38Arg mutation in DAD1 causes apoptosis at restrictive temperatures due to a defect in N-linked glycosylation. To analyze the function of Ost2 in yeast cell death, we constructed Saccharomyces cerevisiae strains expressing Gly58Arg (corresponding to the Gly38Arg mutation in hamster DAD1), Gly86Arg, and Glu113Val mutant Ost2. At elevated temperatures, ost2 mutants arrested growth by decreasing cell viability. Phosphatidylserine exposure, a phenotypic marker of apoptosis in mammalian cells, was found in ost2 mutant cells at 37 degrees C, although DNA fragmentation was not clearly detected. A high concentration of sorbitol compensates for the temperature sensitivity of the ost2 mutant. These results suggest that apoptosis-like cell death in ost2 mutants is caused by the secondary effect of overall reduced protein N-linked glycosylation.     

Multidrug transporter MexB of Pseudomonas aeruginosa: overexpression, purification, and initial structural characterization

Structural and functional characterization of the multidrug transporter, MexB, of Pseudomonas aeruginosa is significantly restricted due to a low yield of approximately 0.1 mg/L of culture from natural sources. To facilitate structural studies of this medically important transporter protein, we developed a large-scale system for expression of the genetically engineered recombinant, MexB, in the Escherichia coli cell. Using the system, the eventual yield of MexB attained was about 10mg/L of culture. The optimized purification protocol in the presence of dodecyl beta-D-maltoside allowed isolation of highly homogeneous MexB. The oligomeric state of the protein in detergent solution has been characterized to verify that the native state of the purified protein has been preserved. The molecular mass of the protein-detergent complex was found to be 380-450kDa. The MexB-dodecyl beta-d-maltoside mass ratio was determined to be 1.8 +/- 0.05. Taking into account the monomeric MexB molecular mass deduced from its amino acid sequence (112.8 kDa), we concluded that the purified MexB exists as the homotrimer in the surfactant solution. Circular dichroism analysis of MexB showed dominance of the alpha-helix structures. High yield, homogeneity, and stability of MexB position it as a good candidate for structural and functional characterization.     

Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH.

The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide (1), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide-receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1-Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible beta-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first beta-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second beta-turn. Furthermore, the presence of a beta-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH-NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I beta-turn structure is also supported by CD spectral analysis. The cIBR peptide (1) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.     

Pyruvate carboxylase: isolation of the biotin-containing tryptic peptide and the determination of its primary sequency

The biotin-containing tryptic peptides of pyruvate carboxylase from sheep, chicken, and turkey liver mitochondria have been isolated and their primary structures determined. The amino acid sequences of the 19 residue peptides from chicken and turkey are identical and share a common sequence of 14 residues around biocytin with the 24-residue peptide isolated from sheep. The sequences obtained were: residue 1 → 11 Avian: Gly Ala Pro Leu Val Leu Ser Ala Met Biocytin Met Sheep: Gly Gln Pro Leu Val Leu Ser Ala Met Biocytin Met residues 12 → 19 or 24 Avian: Glu Thr Val Val Thr Ala Pro Arg Sheep: Glu Thr Val Val Thr Ser Pro Val Thr Glu Gly Val Arg A sensitive radiochemical assay for biotin was developed based on the tight binding of biotin by avidin. The ability of zinc sulfate to precipitate, without dissociating, the avidin-biotin complex provided a convenient procedure for separating free and bound biotin, and hence, for back-titrating a standard amount of avidin with [¹⁴C]biotin.     

Enhanced activity of human serotonin transporter variants associated with autism

Rare, functional, non-synonymous variants in the human serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT) gene (SLC6A4) have been identified in both autism and obsessive-compulsive disorder (OCD). Within autism, rare hSERT coding variants associate with rigid-compulsive traits, suggesting both phenotypic overlap with OCD and a shared relationship with disrupted 5-HT signalling. Here, we document functional perturbations of three of these variants: Ile425Leu; Phe465Leu; and Leu550Val. In transiently transfected HeLa cells, the three variants confer a gain of 5-HT transport phenotype. Specifically, enhanced SERT activity was also observed in lymphoblastoid lines derived from mutation carriers. In contrast to previously characterized Gly56Ala, where increased transport activity derives from catalytic activation, the three novel variants exhibit elevated surface density as revealed through both surface antagonist-binding and biotinylation studies. Unlike Gly56Ala, mutants Ile425Leu, Phe465Leu and Leu550Val retain a capacity for acute PKG and p38 MAPK regulation. However, both Gly56Ala and Ile425Leu demonstrate markedly reduced sensitivity to PP2A antagonists, suggesting that deficits in trafficking and catalytic modulation may derive from a common basis in perturbed phosphatase regulation. When expressed stably from the same genomic locus in CHO cells, both Gly56Ala and Ile425Leu display catalytic activation, accompanied by a striking loss of SERT protein.     

A mutation in the lactose permease of Escherichia coli that decreases conformational flexibility and increases protein stability

Lactose permease with Cys154 --> Gly (helix V) binds substrate with high affinity but catalyzes little or no transport. The purified, detergent-solubilized mutant protein exhibits much greater thermal stability than the wild type and little tendency to aggregate. Stabilization is also observed in vivo with an unstable mutant that is expressed at significantly higher levels when the Cys154 --> Gly mutation is introduced. In addition, ligand-induced conformational changes are markedly reduced or abolished by the Cys154 --> Gly mutation: (i) Although the fluorescence of purified single Trp33 (helix I) permease is enhanced by ligand binding, introduction of the Cys154 --> Gly mutation abolishes the effect. (ii) The rate of 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) labeling of permease with a single Cys residue in place of Val331 (helix X) is increased in the presence of ligand but reduced when the Cys154 --> Gly mutation is present. (iii) Fluorescence emission intensity of MIANS-labeled single Cys331 permease is enhanced and blue shifted in the Cys154 --> Gly mutant background, indicating that the latter mutation causes position 331 to become exposed to a less polar environment. The results indicate that the Cys154 --> Gly mutation causes a more compact structure and decreased conformational flexibility, an alteration that specifically blocks the structural changes necessary for substrate translocation with little or no effect on ligand binding.     

Conservation and divergence on plant seed 11S globulins based on crystal structures

The crystal structures of two pro-11S globulins namely: rapeseed procruciferin and pea prolegumin are presented here. We have extensively compared them with the other known structures of plant seed 11S and 7S globulins. In general, the disordered regions in the crystal structures among the 11S globulins correspond to their five variable regions. Variable region III of procruciferin is relatively short and is in a loop conformation. This region is highly disordered in other pro-11S globulin crystals. Local helical and strand variations also occur across the group despite general structure conservation. We showed how these variations may alter specific physicochemical, functional and physiological properties. Aliphatic hydrophobic residues on the molecular surface correlate well with T_m values of the globulins. We also considered other structural features that were reported to influence thermal stability but no definite conclusion was drawn since each factor has additive or subtractive effect. Comparison between proA3B4 and mature A3B4 revealed an increase in r.m.s.d. values near variable regions II and IV. Both regions are on the IE face. Secondary structure based alignment of 11S and 7S globulins revealed 16 identical residues. Based on proA3B4 sequence, Pro60, Gly128, Phe163, Phe208, Leu213, Leu227, Ile237, Pro382, Val404, Pro425 and Val 466 are involved in trimer formation and stabilization. Gly28, Gly74, Asp135, Gly349 and Gly397 are involved in correct globular folding.     

Conformational aspects of the biological effect of functional fragments of the p21ras oncoprotein family. 2. Fragment 1-16, various sequences]

The conformational analysis data on active ([Val12-Gly13], [Asp12-Gly13] and [Gly12-Asp13]) and passive ([Gly12-Gly13] and [Pro12-Gly13]) modifications of the p21ras family oncoproteins are presented. The activating amino acid substitutions are shown to be accompanied by essential changes in the secondary structure, resulted in the 9-16 fragment spiralization. The spatial structure of the 1-9 fragment does not vary for all the predominant forms of the active and passive analogues. The results of the conformational analysis have been used for studying the structural-functional relationships.     

Impact of retinal disease-associated RPE65 mutations on retinoid isomerization

Pathogenic mutations in the RPE65 gene are associated with a spectrum of congenital blinding diseases in humans. We evaluated changes in the promoter region, coding regions, and exon/intron junctions of the RPE65 gene by direct sequencing of DNA from 36 patients affected with Leber's congenital amaurosis (LCA), 62 with autosomal recessive retinitis pigmentosa (arRP), and 21 with autosomal dominant/recessive cone-rod dystrophies (CORD). Fifteen different variants were found, of which 6 were novel. Interesting was Gly244Val, a novel mutation close to the catalytic center. To assess the role of this mutation in RPE65 inactivation, we performed detailed biochemical studies of the mutant along with a structural analysis of the 244 amino acid position with respect to amino acids known to be important for RPE65-dependent retinoid isomerization. Bicistronic plasmid expression of the RPE65 Gly244Val mutant and enhanced green fluorescent protein (EGFP) allowed us to document both its instability in cultured cells by cell sorting and immunoblotting methodology and its loss of RPE65-dependent isomerase activity by enzymatic assays. Further insights into the structural requirements for retinoid isomerization by RPE65 were obtained by using the carotenoid oxygenase (ACO) from Synechocystis (PDB accession code 2BIW ) as a structural template to construct a RPE65 homology model and locating all known inactivating mutations including Gly244Val within this model.     

Detection of an activating c-kit mutation by real-time PCR in patients with anaphylaxis

A well-characterised gain-of-function point mutation within exon 17 of the c-kit proto-oncogene known as Asp816Val is present in patients with mastocytosis. Activation of mast cells through this receptor primes them for IgE-dependent activation, and patients with mastocytosis are at increased risk of anaphylaxis. We hypothesised that the Asp816Val mutation is associated with a history of anaphylaxis in the general population. A mismatch amplification real-time PCR assay was developed and validated to test for the Asp816Val mutation. Subjects were recruited to four subject groups: normal non-atopics, atopics without anaphylaxis, food-induced anaphylactics and non-food anaphylactics. Blood samples collected from forty subjects were tested for the presence of Asp816Val. Thirteen subjects were found to carry the mutation; normals (2/9), atopics (2/10), food anaphylactics (5/11) and non-food anaphylactics (4/10). Statistical analysis of the data determined that there was no significant difference between the numbers of subjects found to carry the Asp816Val mutation in each of the groups although a trend towards an increased occurrence in anaphylactics was observed. In summary, the hypothesis that the presence of the Asp816Val mutation is linked to the occurrence of anaphylaxis was not supported, but interestingly, we have shown for the first time Asp816Val may occur more frequently than previously reported within the general population.     

High-resolution NMR studies of fibrinogen-like peptides in solution: resonance assignments and conformational analysis of residues 1-23 of the A alpha chain of human fibrinogen

The proton resonances of the following synthetic linear human fibrinogen-like peptides were completely assigned with two-dimensional NMR techniques in solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp(7)-Phe-Leu-Ala-Glu-Gly(12)-Gly(13)-Gly(14)- Val(15)-Arg(16)-Gly-Pro-Arg-Val-Val-Glu-Arg (F10), Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly(13)-Gly(14)-Val-Arg (F11), and Gly-Pro-Arg-Val-Val-Glu-Arg (F12). No predominant structure was found in the chain segment from Ala(1) to Gly(6) for F10 in both H2O and dimethyl sulfoxide. The previous suggestion that there is a hairpin loop involving residues Gly(12) to Val(15) in the A alpha chain of human fibrinogen is supported by the slow backbone NH exchange rates of Gly(14) and Val(15), by an unusually small NH chemical shift of Val(15), and by strong sequential NOE's involving this region in F10. This local chain fold within residues Asp(7) to Val(20) may place the distant Phe residue near the Arg(16)-Gly(17) peptide bond which is cleaved by thrombin.     

A new approach to gene mutation analysis using "GFP-Display"

The unique behavior of green fluorescent protein (GFP) on SDS-PAGE was applied to the detection of a single amino acid substitution in GFP-tagged polypeptides. This simple detection method using SDS/urea gels was designated GFP-display. The N-terminal 18 or 37 amino acids of K-Ras was used as a model GFP-tagged polypeptide. K-ras exon 1 was fused to a gfp cDNA at each end and expressed in Escherichia coli. Amino acid number 12 of K-Ras (wild type; Gly) was changed to Ser, Arg, Cys, Asp, Ala, or Val, and the mobility shift of the greenish fluorescent bands in the SDS/urea gel was analyzed. These mutants were easily detected by GFP-display; however, detection depended strongly on the urea concentration and electrophoresis temperature. Subsequently, GFP-display was applied to the 36 amino acids encoding human p53 exon 7. Amino acid number 248 (wild type; Arg) was changed to Gly, Trp, Gln, Pro, or Leu, and similar mobility shifts were observed. GFP-display could be coupled with an in vitro translation system. Fluorescent active GFP and GFP-Ras fusion proteins were synthesized within a few hours. GFP-display shows potential as a modern approach to gene mutation analysis at the protein level, and is a useful method for protein engineering studies.