Complete amino acid sequence of murine beta 2-microglobulin: structural evidence for strain-related polymorphism.

Primary structural analyses of beta 3-microglobulin isolated from the tumor cell lines EL4.BU (derived from a C57BL/6 mouse) and C14 (derived from a BALB/c mouse) have revealed the presence of an amino acid difference at position 85 of this molecule. beta 2-Microglobulin isolated from histocompatibility antigens of EL4.BU has alanine at this position, whereas that from C14 has aspartic acid. Determination of the sequence of these molecules has employed radiochemical methodology that was developed in studies of murine histocompatibility antigens. The sequence obtained in this study is: Ile - Gln - Lys - Thr - Pro - Gln - Ile - Gln - Val - Tyr - Ser - Arg - His - Pro - Pro - Glu - Asn - Gly - Lys - Pro - Asn - Ile - Leu - Asn - Cys - Tyr - Val - Thr - Gln - Phe - His - Pro - Pro - His - Ile - Glu - Ile - Gln - Met - Leu - Lys - Asn - Gly - Lys - Lys - Ile Pro - Lys - Val - Glu - Met - Ser - Asp - Met - Ser - Phe - Ser - Lys - Asp - Trp - Ser - Phe - Tyr - Ile - Leu - Ala - His - Thr - Glu - Phe - Thr - Pro - Thr - Glu - Thr - Asp - Thr - Tyr - Ala - Cys - Arg - Val - Lys - His - Ala/Asp - Ser - Met - Ala - Glu - Pro - Lys - Thr - Val - Tyr - Trp - Asp - Arg - Asp - Met. Comparison of the sequence of murine beta 2-microglobulin to the sequences reported for the homologues from man, rabbit, and guinea pig indicate identities of 68%, 66%, and 61%, respectively.     

Molecular biology and clinical manifestation of hereditary factor VII deficiency

Inherited factor VII (FVII) deficiency is a rare autosomal recessive disorder. Mutations and polymorphisms of the FVII gene were characterized in more than 40 unrelated patients with FVII deficiency. Among the 29 different mutations, the most frequent were Ala294 Val, Ala294Val;404delC, IVS7+7, and Val281 Phe. Four novel mutations (IVS2+1G>C, Arg247 Cys, Glu265 Lys, Asp343 His) were detected. The relationships between genotypes of mutations and polymorphisms of the FVII gene, FVII deficiency, and clinical phenotype were investigated. Homozygosity of the Phe4 Leu, IVS4+1G>A, Cys135 Arg, Ala244 Val, and Ala294 Val;404delC and the double heterozygosity of Tyr68 Cys / IVS3-1G>A, Val252 Met / IVS2+5G>T, Val281 Phe / Cys135 Arg, Ala294 Val / Val281 Phe, Ala294 Val;404delC / Val281Phe, Ala294 Val;404delC / Arg152 stop, Ala294Val;404delC / Gln(-35) stop, Ala294 Val / Val252 Met, Ala294 Val / Gly156 Asp, and Thr359 Met / Asp242 His were related to clinical symptoms. Double heterozygotes for Arg247 Cys / IVS2+1G>C, Ala206 Thr / Pro303 Arg, Leu(-20) Pro / Val252 Met as well as IVS7+7 /Ala294 Val, IVS7+7 /Ala206 Thr, and IVS7+7 / Met298 Ile were asymptomatic. The clinical symptomatology is rather poor in correlation with the FVII activity. Concerning the clinical phanotype, a correlation seems to exist between specific mutations and clinical symptoms.     

Sequences of the recA gene and protein.

We have determined the nucleotide sequence of the recA gene of Escherichia coli; this permits the formulation of the primary structure for the recA protein. This structure is consistent with the amino acid composition of the tryptic peptides obtained from the recA protein. The coding region of the recA gene has 1059 base pairs, which specify 352 amino acids. The recA protein has alanine and phenylalanine as its NH2- and COOH-terminal amino acids, respectively, and has the following amino acid composition: Cys3 Asp20 Asn15 Met9 Thr17 Ser20 Glu30 Gln13 Pro10 Gly35 Ala38 Val22 Ile27 Leu31 Tyr7 Phe10 His2Lys27 Trp2 Arg14. Of the three cysteine residues, only two can be alkylated under reducing and denaturing conditions. The molecular weight of the recA polypeptide is 37,842.     

Gastrin-releasing peptide from the intestine of the elasmobranch fish, Scyliorhinus canicula (common dogfish)

The concentration of gastrin-releasing peptide in the intestine of the elasmobranchian fish, Scyliorhinus canicula, measured with an antiserum directed against the COOH-terminal region of porcine gastrin-releasing peptide, was higher than the concentrations measured in mammalian intestines. The immunoreactivity was resolved by gel permeation chromatography into two peaks with the approximate elution volumes of porcine gastrin-releasing peptide and bombesin/neuromedin C. The primary structure of the larger peptide was established as Ala Pro Val Glu Asn Gln Gly Ser Phe Pro Lys Met Phe Pro Arg Ser His (Trp) Ala Val Gly (His Leu Met.NH2). Residues in parentheses are only tentatively assigned. Chromatographic evidence and the presence of the arginyl residue at position 15 in the peptide suggest that the smaller molecular form of gastrin-releasing peptide may be identical to mammalian neuromedin C. Amphibian bombesin was not identified in the dogfish gut.     

Amino acid metabolism during exercise in trained rats: the potential role of carnitine in the metabolic fate of branched-chain amino acids

The influence of endurance training and an acute bout of exercise on plasma concentrations of free amino acids and the intermediates of branched-chain amino acid (BCAA) metabolism were investigated in the rat. Training did not affect the plasma amino acid levels in the resting state. Plasma concentrations of alanine (Ala), aspartic acid (Asp), asparagine (Asn), arginine (Arg), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val) were significantly lower, whereas glutamate (Glu), glycine (Gly), ornithine (Orn), tryptophan (Trp), tyrosine (Tyr), creatinine, urea, and ammonia levels were unchanged, after one hour of treadmill running in the trained rats. Plasma concentration of glutamine (Glu), the branched-chain keto acids (BCKA) and short-chain acyl carnitines were elevated with exercise. Ratios of plasma BCAA/BCKA were dramatically lowered by exercise in the trained rats. A decrease in plasma-free carnitine levels was also observed. These data suggest that amino acid metabolism is enhanced by exercise even in the trained state. BCAA may only be partially metabolized within muscle and some of their carbon skeletons are released into the circulation in forms of BCKA and short-chain acyl carnitines.     

Molecular pathology of haemophilia A in Turkish patients: identification of 36 independent mutations

Haemophilia A is an X-linked recessive bleeding disorder caused by heterogeneous mutations in the factor VIII gene. In an attempt to reveal the molecular pathology of Turkish haemophilia A patients, the coding sequence of the gene, excluding a large portion of exon 14, was amplified from genomic DNA and subjected to denaturing gradient gel electrophoresis prior to DNA sequencing. Fifty-nine haemophilia A patients were included in the study with severe, moderate and mild phenotypes observed in 24, 15 and 16 patients, respectively. Factor VIII activity and clinical phenotypes were not available for four patients. A total of 36 independent mutations were found, with a mutation detection efficacy of 61%. The mutations that were reported for the first time include 20 point mutations, one 8-bp insertion (TCAAGATA) in exon 4 and one large deletion greater than 2.8 kb involving exon 14. The novel point mutations were composed of three nonsense (Ser681Ter, Cys2021Ter and Gln2113Ter), one splicing error (IVS-1G-->A), 15 missense mutations (Lys48Asn; Leu-98Phe; Thr118Ala; Cys248Tyr; Glu456Lys; Asp560Ala; Tyr664Cys; Phe679Leu; Gly691Trp; Asp1769His; Val1857Leu; Gly2026Gln; Arg2163Pro; Asp2288Ala; and Arg2304Leu) and a T deletion in exon 25 that caused a frameshift followed by a stop codon. All missense mutations except Val1857Leu, which maintained a conserved nonpolar R group, occurred at amino acids conserved among four species and were most probably pathogenic. In addition, two sequence changes (IVS3-9C-->T) and (Leu2230Leu) were also detected in patients carrying Val1857Leu and Phe679Leu missense mutations, respectively. Identification of mutation origins in eight sporadic cases revealed an equal sex ratio of mutations.     

Plasma free amino acid profiling of esophageal cancer using high-performance liquid chromatography spectroscopy

AIM: To perform plasma free amino acid (PFAA) profiling of esophageal squamous cell carcinoma (ESCC) patients at different pathological stages and healthy subjects.METHODS: Plasma samples from ESCC patients (n = 51) and healthy control adults (n = 60) were analyzed by high-performance liquid chromatography (HPLC). The ESCC patients included moderate/poorly-differentiation (n = 24), lymph node metastasis (n = 17) and clinical stage > Ib2 (n = 36). Partial least squares discriminant analysis was performed to demonstrate that the PFAA metabolic patterns enabled discrimination between ESCC patients and controls, and the Student t test was applied to assess significant differences in PFAA concentrations between the two groups.RESULTS: There were significant differences in the PFAA profiles between controls and ESCC patients. Compared with healthy controls, the levels of Asp, Glu, Gly, His, Thr, Tau, Ala, Met, Ile, Leu, and Phe were decreased in ESCC patients, but Cys was increased. There exists a strong correlation between PFAA profiles and clinicopathological characteristics in ESCC patients. The levels of many PFAAs (i.e., Glu, Asp, Ser, Gly, Tau, Ala, Tyr, Val, Ile, and Leu) were related to pathological grading, lymph node metastasis, and ESCC clinical stage. Very good discrimination between ESCC patients and control subjects was achieved by multivariate modeling of plasma profiles.CONCLUSION: HPLC-based plasma profiling analysis was shown to be an effective approach to differentiate between ESCC patients and controls. PFAA profiles may have potential value for screening or diagnosing ESCC.     

Usefulness of MALDUTOF mass spectrometry of immunoprecipitated serum variant transthyretin in the diagnosis of familial amyloid polyneuropathy

A matrix-assisted laser desorption ionization/time-of flight (MALDI/TOF) mass spectrometry (MS) system was used io detect variant transthyretin (TTR) in immunoprecipitated serum TTR molecules obtained from 6 patients with familial amyloid polyneuropathy (FAP) who were already proven not to have ATTR Val 30Met. This simple and quick method showed six different patterns of mass spectra of TTR-related immunoprecipitates from these patients, and in each patient the clearly identified characteristic doublet-shaped ion peaks consisted of normal and variant TTR apart from each other peak with a mass difference between them. DNA sequencing confirmed that the patterns of variant TTR corresponded respectively to ATTR Val30Leu, ATTR Phe33Val, ATTR Asp38Ala, ATTR Ser50Arg, ATTR Ala97Gly and ATTR Ala97Ser. ATTR Asp38Ala and ATTR Ala97Ser are previously unknown variants of TTR leading to the development of FAP. ATTR Phe33Val was found in a Chinese FAP patient and ATTR Ala97Ser in a Taiwanese. Serum analysis using immunoprecipitation and MALDI/TOF MS system can provide useful information when investigating FAP patients with diverse types of variant TTR.     

Usefulness of MALDI/TOF mass spectrometry of immunoprecipitated serum variant transthyretin in the diagnosis of familial amyloid polyneuropathy.

A matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometry (MS) system was used to detect variant transthyretin (TTR) in immunoprecipitated serum TTR molecules obtained from 6 patients with familial amyloid polyneuropathy (FAP) who were already proven not to have ATTR Val30Met. This simple and quick method showed six different patterns of mass spectra of TTR-related immunoprecipitates from these patients, and in each patient the clearly identified characteristic doublet-shaped ion peaks consisted of normal and variant TTR apart from each other peak with a mass difference between them. DNA sequencing confirmed that the patterns of variant TTR corresponded respectively to ATTR Val30Leu, ATTR Phe33Val, ATTR Asp38Ala, ATTR Ser50Arg, ATTR Ala97Gly and ATTR Ala97Ser. ATTR Asp38Ala and ATTR Ala97Ser are previously unknown variants of TTR leading to the development of FAP. ATTR Phe33Val was found in a Chinese FAP patient and ATTR Ala97Ser in a Taiwanese. Serum analysis using immunoprecipitation and MALDI/TOF MS system can provide useful information when investigating FAP patients with diverse types of variant TTR.     

Leucine and tissue distribution of bulky and small neutral amino acids in rats: Dissociation between transport and insulin-mediated effects

Summary The mechanism of the observed decrease in the plasma concentration of several amino acids in the presence of high levels of Leu has remained unexplained. In the present study a decrease in the plasma concentration of Ile, Val, Phe, Tyr, Met, Ala, Pro and Gly was observed after the intraperitoneal injection of Leu to weanling rats. Decreases in net intracellular concentrations in muscle accompanied the decrease in plasma of all of these amino acids except Pro and Gly. An increase in the distribution ratio muscle/plasma was observed exclusively for Gly after administration of Leu or of a non-insulinogenic transport system L analogue. Diazoxide suppressed the Leu-induced decreases in plasma and muscle intracellular concentrations of Ile and Val as well as of Pro in plasma. An increase in the distribution ratio liver/plasma was observed for Pro and Gly in the absence but not in the presence of diazoxide. All the above changes were statistically significant. Hence insulin probably mediates Leu effects, promoting an increased utilization of Ile and Val in muscle and of Pro in liver. A more direct effect of Leu appears to be involved in the apparent increased utilization of Phe, Tyr and Ala in the same tissue. Gly depletion in plasma can be explained by its trapping by inhibitory action of Leu on the exodus of Gly through transport system L.     

Coenzyme specificity in fungal 17beta-hydroxysteroid dehydrogenase

The 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus is an NADP(H)-dependent member of the short-chain dehydrogenase/reductase superfamily (SDR) that belongs to the cP1 classical subfamily. Here, we have created several mutants by site-directed mutagenesis, and through these we have studied the amino acid residues that are responsible for coenzyme binding and specificity. The Thr202Val and Thr202Ile mutants were inactive, thus confirming the importance of Thr202 for the appropriate orientation of the coenzyme that enables the hydride transfer. The Ala50Arg and Asn51Arg mutants had increased rates of NADPH dissociation, and thus an enhanced substrate oxidation with NADP+, while the Asn51Arg mutant also showed an increased rate of NADP+ dissociation, and thus an enhanced substrate reduction with NADPH. Addition of a negatively-charged amino acid residue at the first position after the second beta-strand (Tyr49Asp) affected the coenzyme specificity and turned the enzyme into an NAD+-dependent oxidase resembling the cD1d subfamily members.     

Structural and functional characterization of novel F7 mutations identified in Chinese factor VII-deficient patients

Hereditary factor VII (FVII) deficiency is a rare recessive bleeding disorder with an estimated prevalence of 1/500 000. We had investigated 50 unrelated Chinese patients with FVII deficiency and identified, in total, 25 mutations, including 18 missense mutations and 5 splicing mutations, on the F7 gene. The nucleotide transition c.1224T>G (p.His408Gln) in exon 9 constitutes a hotspot of mutation, with 19 patients harbouring this genetic variance. Few patients were homozygous or compound heterozygous for deleterious mutations, such as non-sense mutations, large insertion or deletions, indicating that complete deficiency of FVII may not be compatible with life. The eight novel mutations identified in the study, including one small deletion (p.Glu49GlyfsTer101), three type I missense mutations, p.Cys238Phe, p.Gly420Asp, p.Ala252Val and four type II missense mutations, p.Val336Met, p.Ser342Gly, p.Gly432Ser and p.Ile213Asn, were further analysed by in vitro expression and functional studies. The laboratory phenotype and structural analysis confirmed the functional consequence of p.Ile213Asn mutation involving cleavage and activation site. The molecular dynamic simulations and binding energy calculations along with functional probing of p.Gly432Ser mutation revealed the critical role of residue Gly432 in the binding between activated factor VII (factor VIIa) and tissue factor.     

A beta-MSH in the pituitary gland of the spotted dogfish (Scyliorhinus canicula): isolation and structure

A β-type MSH has been isolated from the pituitary of the common dogfish (Scyliorhinus canicula). It differs from all MSH peptides so far isolated in that glycine replaces glutamic acid in the so-called heptapeptide core. By conventional methods the sequence was deduced to be Asx.Glx.Ile.Asx.Tyr.Lys.Met.Gly.His.Phe.Arg.Trp.Gly.Ala.Pro.Met.Asp.Lys.     

The hyperglycemic neuropeptide of the terrestrial isopod, Porcellio dilatatus. I. Isolation and characterization

From isolated sinus glands of Porcellio dilatatus, a hyperglycemic neuropeptide (CHH) was purified by means of a single, two-step purification procedure which consisted of gel chromatography on Sephadex G-50, followed by high-performance liquid chromatography. The 5800- to 6100-Da peptide contains 50-52 amino acids residues. The amino acid composition is (Pro, Ile)1; His1-2; (Thr, Ser, Gly, Met, Tyr)2; (Val, Phe, Lys)3; (Ala, 1/2Cys, Leu, Arg)4; Glx5; Asx7; Trp, n.d. The amino acid composition differs from that of two decapod CHHs analyzed thus far. The N-terminus is blocked. The neuropeptide exhibits little or no interspecific hyperglycemic effect in the brachyuran, Uca pugilator, and its cross-reactivity and potency in the RIA for Carcinus-CHH is very low.     

莫西沙星与左氧氟沙星对结核分枝杆菌的交叉耐药性研究

目的探讨莫西沙星（MXF）与左氧氟沙星(LVF)对结核分枝杆菌的交叉耐药性及耐药基因突变位点,为临床用药提供理论依据。方法选取结核分枝杆菌标准株H₃₇Rv和73株左氧氟沙星耐药的耐多药结核分枝杆菌（MDR-TB）临床分离株以及5株对左氟沙星敏感的MDR-TB临床分离株,以试管二倍稀释法采用7H9培养基,测定以上菌株对MXF和LVF的MIC;通过直接测序法测定gyrA基因喹诺酮类药物耐药决定区（QRDR）320bp的基因突变位点。结果MXF的MIC比LVF低4～8倍;H₃₇Rv未发生gyrA基因突变;78株MDR-TB临床分离株均有Ser95Thr突变;73株LVF耐药的MDR-TB菌株中,35株（47.94%）对LVF低耐药（MIC为1～8μg/ml）的菌株和31株（42.46％）对LVF高耐药株中,除4株只有Ser95Thr突变外,其他菌株同时有Ala90Val、Asp94His、Asp94Asn、Asp94Gly、Asp94Ala、Asp94Tyr突变。结论莫西沙星较左氧氟沙星的抗结核活性强4～8倍,为不完全交叉耐药;gyrA基因90位和94位突变与MDR-TB菌株对LVF、MXF耐药有关,低耐药与高耐药的突变氨基酸种类有差别,有望将测序作为临床左氟沙星分子药敏试验方法。     

A focus on dominant negative variants in a series of 170 heterozygous FXI-deficient patients

Introduction

Dominant-negative effects have been described for 10 F11 variants in the literature.

Aim

The current study aimed at identifying putative dominant-negative F11 variants.

Material and methods

This research consisted in a retrospective analysis of routine laboratory data.

Results

In a series of 170 patients with moderate/mild factor XI (FXI) deficiencies, we identified heterozygous carriers of previously reported dominant-negative variants (p.Ser243Phe, p.Cys416Tyr, and p.Gly418Val) with FXI activities inconsistent with a dominant-negative effect. Our findings also do not support a dominant-negative effect of p.Gly418Ala. We also identified a set of patients carrying heterozygous variants, among which five out of 11 are novel, with FXI activities suggesting a dominant-negative effect (p.His53Tyr, p.Cys110Gly, p.Cys140Tyr, p.Glu245Lys, p.Trp246Cys, p.Glu315Lys, p.Ile421Thr, p.Trp425Cys, p.Glu565Lys, p.Thr593Met, and p.Trp617Ter). However, for all but two of these variants, individuals with close to half normal FXI coagulant activity (FXI:C) were identified, indicating an inconstant dominant effect.

Conclusion

Our data show that for some F11 variants recognized has having dominant-negative effects, such effects actually do not occur in many individuals. The present data suggest that for these patients, the intracellular quality control mechanisms eliminate the variant monomeric polypeptide before homodimer assembly, thereby allowing only the wild-type homodimer to assemble and resulting in half normal activities. In contrast, in patients with markedly decreased activities, some mutant polypeptides might escape this first quality control. In turn, assembly of heterodimeric molecules as well as mutant homodimers would result in activities closer to 1:4 of FXI:C normal range.     

结核分枝杆菌对左氧氟沙星与莫西沙星的交叉耐药性及gyrA和gyrB基因突变分析

目的研究结核分枝杆菌(Mycobacterium tuberculosis,MTB)对左氧氟沙星(levofloxacin,LVF)与莫西沙星(moxifloxacin,MXF)的交叉耐药性,分析gyrA和gyrB基因突变位点分布及突变位点与耐药水平的关系。方法采用改良罗氏培养基检测MTB标准菌株H37Rv、66株LVF耐药和55株LVF敏感的MTB临床分离菌株LVF和MXF的最低抑菌浓度(minimal inhibition concentra-tion,MIC)。通过PCR直接测序法测定gyrA和gyrB耐药基因片段。结果 MXF的MIC比LVF低2~4倍,MXF抗MTB菌株的活性是LVF的2~4倍。MTB标准菌株H37Rv未见gyrA和gyrB基因突变。66株LVF耐药和55株LVF敏感菌株均存在gyrA AGC95ACC(Ser→Thr)突变;55株LVF敏感菌株gyrA和gyrB基因未见其他突变。66株LVF耐药菌株中,40株(60.6%)gyrAGAC94(AAC或GGC或GCC或CAC或TAC)(Asp→Asn或Gly或Ala或His或Tyr)突变;19株(28.8%)gyrA GCG90GTG(Ala→Val)和1株(1.5%)gyrA GCG90AAG(Ala→Lys)(未见报导)双碱基突变;3株(4.6%)gyrA TCG91CCG(Ser→Pro)突变;1株(1.5%)gyrB GAC500AAC(Asp→Asn)突变;2株(3.0%)呈gyrA GAC94(AAC或GCC)(Asp→Asn或Ala)与gyrB GGG551AGG(Gly→Arg)(未见报导)双位点突变。gyrA GAC94(AAC或GGC)(Asp→Asn或Gly)突变引起FQs药物较高水平耐药;GAC94GCC(Asp→Ala)和GCG90GTG(Ala→Val)突变引起FQs药物较低水平耐药。结论 LVF与MXF之间存在交叉耐药,MXF MIC随LVF MIC增高而增高,但耐药水平不同。GyrA基因突变位点可能与耐药水平有关,有可能根据基因突变的位点分析耐药水平。     

Antigenicity of putative phospholipid membrane-binding residues in factor VIII

Most inhibitory antibodies to human factor VIII (fVIII) bind toepitopes in the A2, ap-A3, or C2 domains. The anticoagulant action of antibodies to the C2 domain is due to inhibition of bindingof fVIII to phospholipid. The x-ray structure of the human fVIII C2domain shows a putative hydrophobic, 3-prong, phospholipid membrane-binding site consisting of Met2199/Phe2200, Val2223, andLeu2251/Leu2252. Additionally, Lys2227, near Val2223, is part ofa ring of positively charged residues that may contribute to electrostatic interaction of fVIII with negatively chargedphosphatidylserine. In this study, 8 active mutants of human fVIII(Met2199Ile, Leu2252Phe, Phe2200Leu, Val2223Ala, Lys2227Glu,Met2199Ile/Phe2200Leu, Val2223Ala/Lys2227Glu, andMet2199Ile/Phe2200Leu/Val2223Ala/Lys2227Glu), which were constructed onthe basis of differences between human, porcine, murine, and caninefVIII at proposed phospholipid binding sites, were expressed. Theantigenicity of the mutants toward 5 C2-specific polyclonal humanantibodies was measured by using the Bethesda assay. A human monoclonalanti-C2 antibody, BO2C11, and a murine C2-specific monoclonal antibody,NMC VIII-5, were also included in the analysis. In comparison withwild-type, B-domainless fVIII, the Met2199Ile, Phe2200Leu, and Leu2252single mutants had lower antigenicity toward most of the inhibitors. Incontrast, the Val2223Ala and Lys2227Glu mutants usually showedincreased antigenicity. These results suggest that C2 inhibitorsfrequently target the Met2199/Phe2200 and Leu2251/Leu2252 -hairpinsand are consistent with the hypothesis that these residues participatein binding to phospholipid membranes. In contrast, Val2223 and Lys2227may oppose antibody binding sterically or through stabilization of alow-affinity membrane-binding conformation of the C2 domain.     

Destabilization of osteogenesis imperfecta collagen-like model peptides correlates with the identity of the residue replacing glycine

Mutations resulting in replacement of one obligate Gly residue within the repeating (Gly-Xaa-Yaa)(n) triplet pattern of the collagen type I triple helix are the major cause of osteogenesis imperfecta (OI). Phenotypes of OI involve fragile bones and range from mild to perinatal lethal. In this study, host-guest triple-helical peptides of the form acetyl-(Gly-Pro-Hyp)(3)-Zaa-Pro-Hyp-(Gly-Pro-Hyp)(4)-Gly-Gly-amide are used to isolate the influence of the residue replacing Gly on triple-helix stability, with Zaa = Gly, Ala, Arg, Asp, Glu, Cys, Ser, or Val. Any substitution for Zaa = Gly (melting temperature, T(m) = 45 degrees C) results in a dramatic destabilization of the triple helix. For Ala and Ser, T(m) decreases to approximately 10 degrees C, and for the Arg-, Val-, Glu-, and Asp-containing peptides, T(m) < 0 degrees C. A Gly --> Cys replacement results in T(m) < 0 degrees C under reducing conditions but shows a broad transition (T(m) approximately 19 degrees C) in an oxidizing environment. Addition of trimethylamine N-oxide increases T(m) by approximately 5 degrees C per 1 M trimethylamine N-oxide, resulting in stable triple-helix formation for all peptides and allowing comparison of relative stabilities. The order of disruption of different Gly replacements in these peptides can be represented as Ala 相似文献