Gln Substitution Research Articles

Fumaric aciduria: Mild phenotype in a 8‐year‐old girl with novel mutations

Fumaric aciduria is a rare, autosomal recessive disorder caused by deficient activity of fumarate hydratase (FH). Common clinical features are hypotonia, failure to thrive, severe psychomotor retardation and seizures. Facial dysmorphism and brain malformations are frequent. Recently, some FH gene mutations have been associated with inherited cutaneous and uterine leiomyomas and papillary renal cell cancer. Our patient had a relatively mild phenotype, a previously not reported genotype and familial tumour predisposition. The mother and grandmother had uterine myomas. The paternal grandfather and his two brothers died from lung and laryngeal cancers. The pregnancy was complicated by bleeding and intrauterine growth retardation. Delivery was after 35 weeks, with normal Apgar score. The girl was hypotonic since birth. At age 2 months the parents noticed short apnoeic crises. She could sit at age 1.5 years, and walk with assistance at 4 years. At age 8 years highly increased excretion of fumaric acid was found twice (217 and 445 mmol/mol creatinine). Shortly before that the girl started to have leg and arm spasms. Grand mal seizures occurred twice. Facial dysmorphism included depressed nasal bridge, anteverted ears, hypertelorism and microcephaly. Speech was limited to few disyllables. She was atactic with spastic paraparesis. Brain MRI showed slight ventriculomegaly, white-matter atrophy and hypoplasia of corpus callosum. Activity of FH in fibroblasts was 1.9 nmol/min/mg protein (controls 40-80). Analysis of the FH gene revealed the maternally derived c.1029_1031delAGT mutation, resulting in Val deletion and substitution of Gln by His, and paternally derived c.976C > T mutation, resulting in substitution of Pro by Ser.

Determinants of the Dual Cofactor Specificity and Substrate Cooperativity of the Human Mitochondrial NAD(P)+-dependent Malic Enzyme: FUNCTIONAL ROLES OF GLUTAMINE 362

The human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys362 in the pigeon cytosolic NADP+-dependent malic enzyme has remarkable effects on the binding of NADP+ to the enzyme and on the catalytic power of the enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825). In this study, we investigate the important role of Gln362 in the transformation of cofactor specificity from NAD+ to NADP+ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD+ to NADP+. The Km(NADP) and kcat(NADP) values for this mutant were reduced by 4-6-fold and increased by 5-10-fold, respectively, compared with those for the wild-type enzyme. Furthermore, up to a 2-fold reduction in Km(NADP)/Km(NAD) and elevation of kcat(NADP)/kcat(NAD) were observed for the Q362K enzyme. Mutation of Gln362 to Ala or Asn did not shift its cofactor preference. The Km(NADP)/Km(NAD) and kcat(NADP)/kcat(NAD) values for Q362A and Q362N were comparable with those for the wild-type enzyme. The DeltaG values for Q362A and Q362N with either NAD+ or NADP+ were positive, indicating that substitution of Gln with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gln362 to Lys because the sigmoidal phenomenon appearing in the wild-type enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gln362 mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD+ specificity. In this study, we provide clear evidence that the single mutation of Gln362 to Lys in human m-NAD-ME changes it to an NADP+-dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP+-specific.

Comparison of Repetitive Sequences Derived from High Molecular Weight Subunits of Wheat Glutenin, an Elastomeric Plant Protein

A strategy has been developed to create repetitive peptides incorporating substitutions in the PGQGQQGYYPTSLQQ consensus repeat sequence of high molecular weight subunits in order to investigate natural sequence variations in elastomeric proteins of wheat gluten. After introduction of glutamic and aspartic acid residues, the peptide behaved similarly to the unmodified form at low pH, but became readily water soluble at pH > 6. Substitution of Gln for Leu at position 13 resulted in only small changes to the secondary structure of the water-insoluble peptides, as did Tyr8His and Thr11Ala. The effects of proline substitutions depended on their location: Leu13Pro substitution had little effect on solubility and structure, but Gln6Pro substitution resulted in dramatic changes. Peptides with two Gln6Pro substitutions had similar properties to the water-insoluble parental peptide, but those with 6 or 10 substitutions were readily soluble. The results indicated that specific sequences influence noncovalent intermolecular interactions in wheat gluten proteins.

Catalysis of individual steps of protein splicing of the Pyrococcus abyssi PolII intein

Protein splicing involves the excision of an intervening polypeptide, the intein, from flanking polypeptides, the exteins, concomitant with the specific ligation of the exteins. The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be over-expressed and purified as an unspliced precursor. This allows for a detailed, in vitro kinetic analysis of the individual steps of protein splicing. We have studied the influence of a conserved intein TXXH motif and of the flanking C-terminal residue of the N-extein on the rate and extent of the first step of protein splicing, an amide-thioester rearrangement of the peptide bond linking the N-extein and intein. We also show that the second step, a trans-thioesterification that results in a branched thioester intermediate, is rapidly reversible with respect to formation of the linear thioester. The third step, Gln cyclization coupled to cleavage of the peptide bond linking the intein and C-extein, is rate determining, and its rate and extent are influenced by two conserved intein His residues. The rate of this step is greatly increased upon substitution of Gln with the highly conserved Asn. Our mechanistic work suggests how the intein can catalyze three distinct chemical reactions in proper sequence, which results in a splicing reaction in place of unproductive side-reactions. This material is based upon work supported by the National Science Foundation (Grants DBI-0320824 and MCB/CAREER-0447647) and by the Research Corporation.

Association of Polymorphisms in ERCC2 Gene with Non-Familial Thyroid Cancer Risk

The ERCC2 protein is an evolutionary conserved ATP-dependent helicase that is associated with a TFIIH transcription factor complex and plays an important role in nucleotide excision repair. Mutations in this gene are responsible for xeroderma pigmentosum and also for Cocayne syndrome and trichothiodystrophy. Several single nucleotide polymorphisms have been identified in the ERCC2 locus. Among them, a G23591A polymorphism in the codon 312 results in an Asp --> Asn substitution in a conserved region and a A35931C polymorphism in the codon 751 results in a Lys --> Gln substitution. Because these polymorphisms have been associated with an increased risk for several types of cancers, we carried out an hospital based case-control study in a Caucasian Portuguese population to evaluate the potential role of these polymorphisms on the individual susceptibility to thyroid cancer. The results obtained did not reveal a significant association between each individual polymorphism studied (G23591A and A35931C) and an increased thyroid cancer risk, but individuals homozygous for non-wild-type variants are overrepresented in patients group. The evaluation of the different haplotypes generated by these polymorphisms showed that individuals simultaneously homozygous for rare variants of both polymorphisms have an increased risk for thyroid cancer [adjusted odds ratio (OR) 3.084; 95% confidence interval (95% CI), 1.347-7.061; P = 0.008] and for papillary thyroid-type tumors (adjusted OR, 2.997; 95% CI, 1.235-7.272; P = 0.015) but not for follicular thyroid-type tumors. These results suggest that genetic polymorphisms in this gene might be associated with individual susceptibility towards thyroid cancer, mainly papillary-type tumors, but larger studies are required to confirm these results.

Acidic residues emulate a phosphorylation switch to enhance the activity of rat hepatic neutral cytosolic cholesterol esterase

Site-directed mutagenesis of rat hepatic neutral cytosolic cholesteryl ester hydrolase (rhncCEH) was used to substitute acidic, basic or neutral amino acid residues for Ser506, required for activation by protein kinase A. The substitution of acidic Asp506 resulted in esterase activities with cholesteryl oleate, p-nitrophenylcaprylate (PNPC) and p-nitrophenylacetate (PNPA) equivalent to those of native rhncCEH with Ser506. The substitution of 2 acidic residues (Asp505/506), emulating the 2 negative charges of phosphoserine, resulted in a 10-fold greater cholesterol esterase activity than that of native rhncCEH, similar to the activity of rhncCEH treated with protein kinase A. In contrast to mutants with Ser506, protein kinase A did not increase the specific activities of mutants with Asp505/506. The substitution of basic (Lys506) or neutral (Asn506) residues abolished activity with cholesteryl oleate but not PNPC or PNPA. The substitution of neutral Gln for basic residues Lys496/Arg503 also abolished cholesterol esterase activity but not PNPC- and PNPA-esterase activities. These structure–activity relationships are modeled by homology with a recently reported crystal structure for the homologous human triacylglycerol hydrolase. The results suggest that the cholesterol esterase activity of carboxylesterases is enhanced by interactions between one or more basic residues on helix α16 (residues 485–503) and acidic groups at residues 505–506 in the adjacent surface loop.

Kinetic Analysis of the Individual Steps of Protein Splicing for the Pyrococcus abyssi PolII Intein

Protein splicing involves the excision of an intervening polypeptide, the intein, from flanking polypeptides, the exteins, concomitant with the specific ligation of the exteins. The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed and purified as an unspliced precursor, which allows for a detailed in vitro kinetic analysis of the individual steps of protein splicing. The first order rate constant for splicing of this intein, which has a non-canonical Gln at its C terminus, is 9.3 x 10(-6) s(-1) at 60 degrees C. The rate constant for splicing increases 3-fold with substitution of Asn for the C-terminal Gln. The pseudo first order rate constant of dithiothreitol-dependent N-terminal cleavage is 1 x 10(-4) s(-1). The first order rate constant of C-terminal cleavage is 1.2 x 10(-5) s(-1) with Gln at the C-terminal position, 2.8 x 10(-4) s(-1) with Asn, and decreases significantly with mutation of the penultimate His of the intein to Ala. N-terminal cleavage is most efficient between pH 7 and 7.5 and decreases at both more acidic and alkaline pH values, whereas C-terminal cleavage and splicing are both efficient over a broader range of pH values.

Novel aberrant splicings caused by a splice site mutation (IVS1a+5g>a) in F7 gene

Low FVII coagulant activity (FVII:C 8.2%) and antigen level (FVII:Ag 34.1%) in a 46-year-old Chinese male led to a diagnosis of coagulation factor VII (FVII) deficiency. Compound heterozygous mutations were identified in his F 7 gene:a G to A transition in the 5' donor splice site of intron 1a (IVS1a+5g>a) and a T to G transition at the nucleotide position 10961 in exon 8, resulting in a His to Gln substitution at amino acid residue 348. An analysis of ectopic transcripts of F7 in the leukocytes of the patient reveals that the mutation (IVS1a+5g>a) is associated with two novel aberrant patterns of splicing. The predominant alternative transcript removes exon 2, but retains intron 3, which shifts the reading frame and predicts a premature translation termination at the nucleotide positions 2-4 in intron 3. The minor alternative transcript skips both exon 2 and exon 3 (FVII Delta 2, 3), leading to an in-frame deletion of the propeptide and gamma-carboxylated glutamic acid (Gla) domains of mature FVII protein. In vitro expression studies of the alternative transcript FVII Delta 2,3 by transient transfection of HEK 293 cells with PcDNA 3.1(-) expression vector showed that although the mutant protein could be secreted, no pro-coagulation activity was detected. The coexistence of the two abnormal transcripts and a heterozygous mutation His348Gln, explained the patient's phenotype.

In vitro generation and characterisation of an influenza B variant with reduced sensitivity to neuraminidase inhibitors

A contemporary influenza type B virus was passaged in vitro in the presence of increasing concentrations of the neuraminidase inhibitors, zanamivir and oseltamivir carboxylate (0.1–1000 μM over nine passages). After the fifth passage in the presence of zanamivir (10 μM), the virus acquired a Glu 119 Asp neuraminidase mutation (influenza A N2 subtype numbering) in the enzyme active site. After a further three passages, in which growth occurred in 100 μM of zanamivir, a Gln 218 Lys mutation (A (H3) numbering) in the HA1 domain of the haemagglutinin was found. In a fluorescence-based neuraminidase inhibition assay, viruses with the Glu 119 Asp NA mutation had a 32,000-fold reduction in sensitivity to the NA inhibitor zanamivir compared to the wild-type virus, while the mutation resulted in a 105-fold reduction in sensitivity to oseltamivir carboxylate. Viruses grown in the presence of 1000 μM oseltamivir carboxylate did not acquire any neuraminidase mutations but did have a His 103 Gln substitution (A (H3) numbering) in the HA1 region of the haemagglutinin which was demonstrated to significantly reduce receptor binding strength in vitro. Tissue culture assays demonstrated that the HA mutation caused a seven-fold reduction in sensitivity to oseltamivir carboxylate, and a 90-fold reduction in sensitivity to zanamivir.

M.465 Molecular genetic analysis of familial hyperchoelsterolemia: Novel LDL mutations in Spanish population

Familial defective apolipoprotein (apo) B-100 (FDB) is caused by R3500Q mutation of the apo B gene resulting in decreased binding of LDL to the LDL receptor. Two other apo B mutations, R3500W and R3531C, affecting binding are known to date. We screened the apo B gene segment around codon 3500 by heteroduplex analysis and single strand conformation polymorphism (SSCP) analysis in a total of 373 hyperlipidemic individuals. Two single-base mutations were detected and confirmed by DNA sequencing. One mutation, ACA3528→ACG change, resulted in degenerate codon with no amino acid substitution. The other mutation, CGG3500→CAG mutation, resulted in an Arg3500→Gln substitution (R3500Q). The prevalence of heterozygote in this selected population was 0.3% (95% confidence interval, 0.01–1.5%) for the R3500Q mutation, and 2.4% (95% confidence interval, 1.1–4.5%) for the previously described R3500W mutation. The results suggest that the R3500Q mutation is not a significant factor contributing to moderate hypercholesterolemia in Chinese (P=0.027). Family studies of the R3500Q carrier revealed a further two individuals heterozygous for the mutation, both of whom were hypercholesterolemic. Analysis of the R3500Q allele using six diallelic markers and the 3′HVR marker revealed a haplotype which was the same as that reported in a Chinese American but differed from that reported in a Chinese Canadian. Our data support limited multiple recurrent origins for R3500Q in Chinese population.

Differences in malate dehydrogenases from the obligately piezophilic deep-sea bacterium Moritella sp. strain 2D2 and the psychrophilic bacterium Moritella sp. strain 5710

The gene encoding malate dehydrogenase (MDH) of the obligately piezophilic deep-sea bacterium Moritella sp. strain 2D2 was cloned and sequenced. There were two positions [close to the active site (Ala-180) and in the subunit interaction site (His-229)] with 2D2-specific substitutions. The MDH genes of strain 2D2 and a psychrophilic bacterium Moritella sp. strain 5710 exhibiting the highest sequence similarity were overexpressed in Escherichia coli. The 2D2 MDH was more heat-stable than the 5710 MDH. The apparent K m value at 62.1 MPa for NADH of the 2D2 MDH was higher than that of the 5710 MDH. The 2D2 MDH in which a His–Gln substitution was introduced at position 229 decreased the thermal stability and K m value at 62.1 MPa. The 5710 MDH that was substituted Gln-229 with His increased the thermal stability and K m value at 62.1 MPa. These results indicate that the His residue at position 229 of the 2D2 MDH may play a role in the thermal stability and the MDH function at high pressure.

DNA repair gene polymorphisms, pre-natal factors and the frequency of somatic mutations in the glycophorin-A gene among healthy newborns

This study investigates variation in somatic mutation frequency, as measured by the glycophorin-A (GPA) somatic mutation assay, in relation to polymorphic variation among 435 newborn babies in DNA repair genes XRCC1, XRCC3 and XRCC4 and gender, parental age, social class and smoking habits. The three polymorphisms under investigation were an Arg→Gln substitution at codon 399 in exon 10 of XRCC1, a Thr→Met substitution at codon 241 in exon 7 of XRCC3 and an Ile→Thr substitution at codon 401 in exon 4 of XRCC4. The study population is an extension of that previously analysed for GPA mutations and XRCC1 polymorphisms. A significant difference was seen in the earlier work in the genotype distribution for the XRCC1 Arg399Gln polymorphism between the main population and the small number with extreme values for NN variant frequency and this was maintained in the larger study group (OR 3.20 [95% CI: 1.16, 8.81]) P=0.043). No such association was seen for XRCC3 or XRCC4 polymorphisms. When adjustments were made for multiple testing, neither N0 nor NN variant frequencies in the main study population were found to be influenced by the polymorphisms in XRCC1, XRCC3, or XRCC4. In addition, neither maternal or paternal smoking, age or social class nor the gender of the offspring were found to affect variant frequencies nor were variant frequencies influenced by any interaction between any of these factors and genotype. It is concluded that the genotypic variation in DNA repair genes examined in this study has no discernable effect on the genesis of the somatic mutations observed at birth.

A single point mutation in Drosophila dihydrofolate reductase confers methotrexate resistance to a transgenic CHO cell line.

Sequence analysis of a cDNA encoding dihydrofolate reductase (DHFR) from a selected methotrexate-resistant Drosophila melanogaster cell line (S3MTX) revealed a substitution of Gln for Leu at position 30. Although the S3MTX cells were approximately 1000 fold more resistant to methotrexate (MTX), the karyotype was similar to the parental line and did not show elongated chromosomes. Furthermore, kinetic analysis of the recombinant enzyme showed a decreased affinity for MTX by the mutant DHFR. To determine if the resistance phenotype could be attributed to the mutant allele, Drosophila Dhfr cDNAs isolated from wild type and S3MTX cells were expressed in Chinese hamster ovary (CHO) cells lacking endogenous DHFR. The heterologous insect DHFRs were functional in transgenic clonal cell lines, showing approximately 400-fold greater MTX resistance in the cell line transfected with the mutant Dhfr than the wild type Dhfr. Resistance to other antifolates in the CHO cells was consistent with the drug sensitivities seen in the respective Drosophila cell lines. ELevated Levels of Dhfr transcript and DHFR in transgenic CHO cells bearing the mutant cDNA were not seen. Taken together, these results demonstrate that a single substitution in Drosophila DHFR alone can confer Levels of MTX resistance comparable with that observed after considerable gene amplification in mammalian cells.

A new role of Pro-73 of p47 phox in the activation of neutrophil NADPH oxidase

The PX domain of p47 phox is thought to be involved in autoinhibition. However, when the domain was deleted, the ability to activate the phagocyte NADPH oxidase was markedly diminished. We have mutated the proline-rich region of the PX domain and examined the mutants for the ability to activate. Substitution of Gln for Pro-73 of p47 phox (1–286) (P73Q) resulted in a considerably lower activity than the wild type and P73Q had a much lower affinity for the oxidase complex. Whereas, Gln substitution for Pro-76 (P76Q) showed a slightly enhanced activation and the mutant had a slightly higher affinity for the complex than the wild type. Affinity for p67 phox (1–210) was slightly decreased either by P73Q or P76Q. Optimal SDS concentration for the activation was lowered by these mutations. Binding of PX domain with phosphatidylinositol-3,4-bisphosphate was diminished by P73Q mutation. The results in this study suggest that Pro-73 has a role in interaction with the catalytic component cytochrome b 558.

Calmodulin Oxidation and Methionine to Glutamine Substitutions Reveal Methionine Residues Critical for Functional Interaction with Ryanodine Receptor-1

Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) with high affinity, and it may act as a Ca(2+)-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca(2+)-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca(2+)-CaM, as assessed via determinations of [(3)H]ryanodine and [(35)S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca(2+)-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca(2+)-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca(2+) release during oxidative stress.

Role of conserved Asp293 of cytochrome P450 2C9 in substrate recognition and catalytic activity.

Human cytochrome P450 2C9 (CYP2C9) is important in the metabolism of non-steroidal anti-inflammatory compounds such as diclofenac, the antidiabetic agent tolbutamide and other clinically important drugs, many of which are weakly acidic. Multiple sequence alignment of CYPs identified CYP2C9 Asp(293) as corresponding to Asp(301) of CYP2D6, which has been suggested to play a role in the binding of basic substrates to the latter enzyme. Replacement of Asp(293) with Ala (D293A) decreased activity by more than 90%, and led to an approx. 3- to 10-fold increase in K (m) values for the three test substrates tolbutamide, dextromethorphan and diclofenac. Conservative replacement of the carboxyl side chain in a Glu (D293E) mutant produced no significant changes in K (m) values and slight increases in k (cat) values. Changes in regiospecificity were observed for both the Ala and Glu substitutions; low levels of both dextromethorphan O- and N-demethylation were observed in the D293A mutant, whereas increased preference for O-demethylation was observed for the D293E mutant. Expression of constructs coding for Asn (D293N) and Gln (D293Q) substitutions failed to form a P450 correctly. Our analysis suggests a structural role for the carboxyl side chain of Asp(293) in CYP2C9 substrate binding and catalysis. The conservation of an Asp residue in other CYP families in a position equivalent to Asp(293) indicates a common mechanism for maintaining the active-site architecture.

Functional evidence for an intramolecular side chain interaction between residues 6 and 10 of receptor-bound parathyroid hormone analogues.

The N-terminal domain of PTH(1-34) is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated the possibility that the side chains of residues 6 (Gln) and 10 (Gln or Asn) of PTH analogues, which would align on the same face of the predicted alpha-helix, could interact and thereby contribute to the PTH/P1R interaction process. We utilized PTH(1-11), PTH(1-14), and PTH(1-34) analogues substituted with alanine at one or both of these positions and functionally evaluated the peptides in cell lines (HKRK-B7 and HKRK-B28) stably expressing the P1R, as well as in COS-7 cells transiently expressing either the P1R or a P1R construct that lacks the amino-terminal extracellular domain (P1R-DelNt). In HKRK-B7 cells, the single substitutions of Gln(6) --> Ala and Gln(10) --> Ala reduced the cAMP-stimulating potency of [Ala(3),Gln(10),Arg(11)]rPTH(1-11)NH(2) approximately 60- and approximately 2-fold, respectively, whereas the combined Ala(6,10) substitution resulted in a approximately 2-fold gain in potency, relative to the single Ala(6) substitution. Similar effects on P1R-mediated cAMP-signaling potency and P1R-binding affinity were observed for these substitutions in [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]rPTH(1-14)NH(2). Installation of a lactam bridge between the Lys(6) and the Glu(10) side chains of [Ala(3,12),Lys(6),Glu(10),Har(11),Trp(14)]rPTH(1-14)NH(2) increased signaling potency 6-fold, relative to the nonbridged linear analogue. Alanine substitutions at positions 6 and/or 10 of [Tyr(34)]hPTH(1-34)NH(2) did not affect signaling potency nor binding affinity on the intact P1R; however, Ala(6) abolished PTH(1-34) signaling on P1R-DelNt, and this effect was reversed by Ala(10). The overall data support the hypothesis that the N-terminal portion of PTH is alpha-helical when bound to the activation domain of the PTH-1 receptor and they further suggest that intrahelical side chain interactions between residues 6 and 10 of the ligand can contribute to the receptor interaction process.

Identification and characterization of AMPK gamma 3 mutations in the pig.

The RN(-) (rendement Napole, French for Napole yield) phenotype is common in Hampshire pigs and is characterized by a 70% increase in glycogen content in skeletal muscle and large effects on meat characteristics (pH, water content, technological yield and lean meat content). The phenotype is controlled by an autosomal dominant allele designated RN (-). The protein kinase AMP-activated gamma 3 subunit gene, PRKAG3, which encodes the gamma 3 isoform of AMP-activated protein kinase (AMPK), was identified as the causative gene for this phenotype by a pure positional cloning approach. There are now several lines of evidence supporting our interpretation that the RN(-) phenotype is caused by a missense mutation (Arg(200)-->Gln) in PRKAG3. Recent data from another group have revealed the presence of a third functional allele at the PRKAG3 locus, probably caused by a Val(199)-->Ile missense mutation. This allele has opposite effects compared with RN, as it is associated with a low glycogen content. We have confirmed the phenotypic effect of this third allele in a meat-quality study of a Hampshire/Landrace intercross. A physiological characterization of RN(-) carriers and normal pigs showed that the RN(-) pigs utilized glycogen during exercise to the same extent as normal pigs and they showed a significantly faster resynthesis of glycogen after exercise. The results strongly suggest that the Arg(200)-->Gln substitution is not associated with a defect in glycogen degradation, but rather with an increased glucose uptake in skeletal muscle.

Virus-cell fusion inhibitory activity of novel analogue peptides based on the HP (2-20) derived from N-terminus of Helicobacter pylori Ribosomal Protein L1.

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is the antibacterial sequence derived from N-terminus of Helicobacter pylori Ribosomal Protein L1 (RPL1). It has a broad-spectrum microbicidal activity in vitro that is thought to be related to the membrane-disruptive properties of the peptide. Based on the putative membrane-targeted mode of action, we postulated that HP (2-20) might be possessed virus-cell fusion inhibitory activity. To develop the novel virus-cell fusion inhibitory peptides, several analogues with amino acid substitution were designed to increase or decrease only net hydrophobic region. In particular, substitution of Gln and Asp for hydrophobic amino acid, Trp at position 17 and 19 of HP (2-20) (Anal 3) caused a dramatic increase in virus-cell fusion inhibitory activity without hemolytic effect.

Influence of the Protein Environment on the Properties of a Tyrosyl Radical in Reaction Centers from Rhodobacter sphaeroides

The influence of the local environment on the formation of a tyrosyl radical was investigated in modified photosynthetic reaction centers from Rhodobacter sphaeroides. The reaction centers contain a tyrosine residue placed approximately 10 A from a highly oxidizing bacteriochlorophyll dimer. Measurements by both optical and electron paramagnetic resonance spectroscopy revealed spectral features that are assigned as arising primarily from an oxidized bacteriochlorophyll dimer at low pH values and from a tyrosyl radical at high pH values, with a well-defined transition that occurred with a pK(a) of 6.9. A model based on the wild-type structure indicated that the Tyr at M164 is likely to form a hydrogen bond with His M193 and to interact weakly with Glu M173. Substitution of Tyr or Glu for His at M193 increased the pK(a) for the transition from 6.9 to 8.9, while substitution of Gln for His M193 resulted in a higher pK(a) value. Substitution of Glu M173 with Gln resulted in loss of the partial formation of the tyrosyl that occurs in the other mutants at low pH values. The results are interpreted in terms of the ability of the residues to act as proton acceptors for the oxidized tyrosine, with the pK(a) values reflecting those of either the putative proton acceptor or the tyrosine, in accord with general models of amino acid radicals.