NMR study of silk I structure of Bombyx mori silk fibroin with 15N- and 13C-NMR chemical shift contour plots

The metastable state silk I structures of Bombyx mori silk fibroin in the solid state were studied on the basis of ¹⁵N- and ¹³C-nmr chemical shifts of Ala, Ser, and Gly residues. The ¹⁵N cross-polarization magic angle spinning (CP/MAS) nmr spectra of the precipitated fraction after chymotrypsin hydrolysis of B. mori silk fibroin with the silk I and silk II forms were measured to determine the ¹⁵N chemical shifts of Gly, Ala, and Ser residues. For comparison, ¹⁵N CP/MAS nmr chemical shifts of Ala were measured for [¹⁵N] Ala Philosamia cynthia ricini silk fibroin with antiparallel β-sheet and α-helix forms. The ¹³C CP/MAS nmr chemical shifts of Ala, Ser, and Gly residues of B. mori silk fibroin with the silk I and silk II forms, as well as ¹³C CP/MAS nmr chemical shifts of Ala residue of P. c. ricini silk fibroin with β-sheet and α-helix forms, are used for the examination of the silk I structure. Both silk I and α-helix peaks are shifted to a lower field than silk II (β-sheet) for the Cα carbons of the Ala residues, while both Cβ carbon peaks are shifted to higher field. However, the silk I peak of the ¹⁵N nucleus of the Ala residue is shifted to lower field than the silk II peak, but the α-helix peak is shifted to high field. Thus, the difference in the structure between the silk I and α-helix is reflected in a different manner between the ¹³C and ¹⁵N chemical shifts. The Cα and Cβ chemical shift contour plots for Ala and Ser residues, and the Cα plot for the Gly residue, were prepared from the Protein Data Bank data obtained for 12 proteins and used for discussing the silk I structure quantitatively from the conformation-dependent chemical shifts. The plots reported by Le and Oldfield for ¹⁵N chemical shifts were also used for the purpose. All these chemical shift data support Fossey's model (Ala: ϕ = −80°, φ = 150°, Gly: ϕ = −150°, φ = 80°) and do not support Lotz and Keith's model (Ala: ϕ = −104.6°, φ = 112.2°, Gly: ϕ = 79.8°, φ = 49.7° or Ala: ϕ = −124.5°, φ = 88.2°, Gly: ϕ = −49.8°, φ = −76.1°) as the silk I structure. © 1997 John Wiley & Sons, Inc.     

A high-resolution 13C-nmr study of collagenlike polypeptides and collagen fibrils in solid state studied by the cross-polarization–magic angle-spinning method. Manifestation of conformation-dependent 13C chemical shifts and application to conformational characterization

We have recorded high-resolution ¹³C-nmr spectra of collagen fibrils in the solid state by the cross-polarization–magic-angle-spinning(CP–MAS)method and analyzed the spectra with reference to those of collagenlike polypeptides. We used two kinds of model polypeptides to obtain reference ¹³C chemical shifts of major amino acid residues of collagen (Gly, Pro, Ala, and Hyp): the 3₁-helical polypeptides [(Gly)_nII, (Pro)_nII, (Hyp)_n, and (Ala? Gly? Gly)_nII], and the triple-helical polypeptides [(Pro? Gly? Pro)_n and (Pro? Ala? Gly)_n]. Examination of the ¹³C chemical shifts of these polypeptides, together with our previous data, showed that the ¹³C chemical shifts of individual amino acid residues are the same, within experimental error (±0.5 ppm), among different polypeptides with different primary sequences, if the conformations are the same. We found that the ¹³C chemical shifts of Ala residues of the 3₁-helical (Ala? Gly? Gly)_n and triple-helical (Pro? Ala? Gly)_n are significantly displaced, compared with those of the α-helix, β-sheet, and silk I form, and can be utilized as excellent probes to examine conformational features of collagen-like polypeptides. Further, the ¹³C chemical shifts of Gly and Pro residues in the triple-helical polypeptides are substantially displaced from those found in (Gly)_nII and (Pro)_nII of the 3₁-helix, reflecting further conformational change from the 3₁-helix to the supercoiled triple helix. In particular, the ¹³C chemical shifts of Gly C ? O carbons of the triple-helical polypeptides are substantially displaced upfield (4.1–5.1 ppm), with respect to those of the 3₁-helical polypeptides. These displacements are interpreted by that Gly C ? O of the former is not involved in NH …? O ? C hydrogen bonds, while this carbon of the latter is linked by these kinds of hydrogen bonds. On the basis of these ¹³C chemical shifts, as reference data for the collagenlike structure, we were able to assign the ¹³C-nmr peaks of Gly, Ala, Pro, and Hyp residues of collagen fibrils, which are in good agreement with the values expected from the model polypeptides mentioned above. We also discuss a plausible conformational change of collagen fibrils during denaturation.     

Synthesis,conformational study,and spectroscopic characterization of the cyclic Cα,α‐disubstituted glycine 9‐amino‐9‐fluorenecarboxylic acid

A series of terminally blocked peptides (to the pentamer level) from l ‐Ala and the cyclic C^α,α‐disubstituted Gly residue Afc and one Gly/Afc dipeptide have been synthesized by solution method and fully characterized. The molecular structure of the amino acid derivative Boc‐Afc‐OMe and the dipeptide Boc‐Afc‐Gly‐OMe were determined in the crystal state by X‐ray diffraction. In addition, the preferred conformation of all of the model peptides was assessed in deuterochloroform solution by FT‐IR absorption and ¹H‐NMR. The experimental data favour the conclusion that the Afc residue tends to adopt either the fully‐extended (C₅) or a folded/helical structure. In particular, the former conformation is highly populated in solution and is also that found in the crystal state in the two compounds investigated. A comparison with the structural propensities of the strictly related C^α,α‐disubstituted Gly residues Ac₅c and Dϕg is made and the implications for the use of the Afc residue in conformationally constrained analogues of bioactive peptides are briefly examined. A spectroscopic (UV absorption, fluorescence, CD) characterization of this novel aromatic C^α,α‐disubstituted Gly residue is also reported. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Directed evolution of Aspergillus niger glucoamylase to increase thermostability

Using directed evolution and site‐directed mutagenesis, we have isolated a highly thermostable variant of Aspergillus niger glucoamylase (GA), designated CR2‐1 . CR2‐1 includes the previously described mutations Asn20Cys and Ala27Cys (forming a new disulfide bond), Ser30Pro, Thr62Ala, Ser119Pro, Gly137Ala, Thr290Ala, His391Tyr and Ser436Pro. In addition, CR2‐1 includes several new putative thermostable mutations, Val59Ala, Val88Ile, Ser211Pro, Asp293Ala, Thr390Ser, Tyr402Phe and Glu408Lys, identified by directed evolution. CR2‐1 GA has a catalytic efficiency (k_cat/K_m) at 35°C and a specific activity at 50°C similar to that of wild‐type GA. Irreversible inactivation tests indicated that CR2‐1 increases the free energy of thermoinactivation at 80°C by 10 kJ mol^?1 compared with that of wild‐type GA. Thus, CR2‐1 is more thermostable (by 5 kJ mol^?1 at 80°C) than the most thermostable A. niger GA variant previously described, THS8 . In addition, Val59Ala and Glu408Lys were shown to individually increase the thermostability in GA variants by 1 and 2 kJ mol^?1, respectively, at 80°C.     

Kinetic studies on the chymotrypsin Aα-catalyzed hydrolysis of a series of N-acetyl-l-phenylalanyl peptides

A series of N-acetyl-l-phenylalanyl peptides of general formula Ac-Phe-(Gly)_n-NH₂ (n = 0–2) has been synthesized to study the effect of leaving group chain length on the efficiency of chymotrypsin A_α amidase and peptidase activities. The effect upon catalysis of hydrophobic side chains on the leaving group was investigated using similar substrates with one of the glycine residues selectively substituted by an alanine residue as in AcPheAlaNH₂, AcPheAlaGlyNH₂, and AcPheGlyAlaNH₂. Values of k_cat and K_m have been obtained from kinetic measurements at pH 8.00 and 25 °C. The results are shown to be consistent with binding schemes postulated from published model building studies. The catalytic reactions were studied over a range of temperature (15–35 °C) and in each case the Arrhenius law was obeyed. It was thus possible to obtain meaningful values for the thermodynamic functions of activation for the acylation step of the catalytic reaction. The results are shown to confirm the findings of postulated binding schemes but indicate that conclusions drawn from kinetic measurements at a single temperature may sometimes be misleading.     

Bacterial collagen‐binding domain targets undertwisted regions of collagen

Clostridium histolyticum collagenase causes extensive degradation of collagen in connective tissue that results in gas gangrene. The C‐terminal collagen‐binding domain (CBD) of these enzymes is the minimal segment required to bind to a collagen fibril. CBD binds unidirectionally to the undertwisted C‐terminus of triple helical collagen. Here, we examine whether CBD could also target undertwisted regions even in the middle of the triple helix. Collageneous peptides with an additional undertwisted region were synthesized by introducing a Gly → Ala substitution [(POG)_xPOA(POG)_y]₃, where x + y = 9 and x > 3). ¹H–¹⁵N heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) titration studies with ¹⁵N‐labeled CBD demonstrated that the minicollagen binds to a 10 Å wide 25 Å long cleft. Six collagenous peptides each labeled with a nitroxide radical were then titrated with ¹⁵N‐labeled CBD. CBD binds to either the Gly → Ala substitution site or to the C‐terminus of each minicollagen. Small‐angle X‐ray scattering measurements revealed that CBD prefers to bind the Gly → Ala site to the C‐terminus. The HSQC NMR spectra of ¹⁵N‐labeled minicollagen and minicollagen with undertwisted regions were unaffected by the titration of unlabeled CBD. The results imply that CBD binds to the undertwisted region of the minicollagen but does not actively unwind the triple helix.     

Ramachandran analysis of conserved glycyl residues in homologous proteins of known structure

High conservation of glycyl residues in homologous proteins is fairly frequent. It is commonly understood that glycine tends to be highly conserved either because of its unique Ramachandran angles or to avoid steric clash that would arise with a larger side chain. Using a database of aligned 3D structures of homologous proteins we identified conserved Gly in 288 alignment positions from 85 families. Ninety‐six of these alignment positions correspond to conserved Gly residue with (φ, ψ) values allowed for non‐glycyl residues. Reasons for this observation were investigated by in‐silico mutation of these glycyl residues to Ala. We found in 94% of the cases a short contact exists between the C^β atom of the introduced Ala with the atoms which are often distant in the primary structure. This suggests the lack of space even for a short side chain thereby explaining high conservation of glycyl residues even when they adopt (φ, ψ) values allowed for Ala. In 189 alignment positions, the conserved glycyl residues adopt (φ, ψ) values which are disallowed for Ala. In‐silico mutation of these Gly residues to Ala almost always results in steric hindrance involving C^β atom of Ala as one would expect by comparing Ramachandran maps for Ala and Gly. Rare occurrence of the disallowed glycyl conformations even in ultrahigh resolution protein structures are accompanied by short contacts in the crystal structures and such disallowed conformations are not conserved in the homologues. These observations raise the doubt on the accuracy of such glycyl conformations in proteins.     

The impact of β‐azido(or 1‐piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues

The synthesis of new dermorphin analogues is described. The (R)‐alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α‐methyl‐β‐azidoalanine or α‐benzyl‐β‐azido(1‐piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). The most active analogue in this series, Tyr‐(R)‐Ala‐(R)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ and its epimer were analysed by ¹H and ¹³C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C^α in the α‐benzyl‐β‐azidoAla residue in position 3. The (R) configuration led to the formation of a type I β‐turn, whilst switching to the (S) configuration gave rise to an inverse β‐turn of type I′, followed by the formation of a very short β‐sheet. The selectivity of Tyr‐(R)‐Ala‐(R) and (S)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Nonionic but water soluble, [Glycine-Pd-Alanine] and [Glycine-Pd-Valine] complexes. Their synthesis,characterization, antitumor activities and rich DNA/HSA interaction studies

Two novel, neutral and water soluble Pd(II) complexes of formula [Pd(Gly)(Ala)] (1) and [Pd(Gly)(Val)] (2) (Gly, Ala, and Val are anionic forms of glycine, alanine, and valine amino acids, respectively) have been synthesized and characterized by FT-IR, UV–Vis, ¹H-NMR, elemental analysis, and molar conductivity measurement. The data revealed that each amino acid binds to Pd(II) through the nitrogen of –NH₂ and the oxygen of –COO^– groups and acts as a bidentate chelate. These complexes have been assayed against leukemia cells (K₅₆₂) using MTT method. The results indicated that both of the complexes display more cytotoxicity than the well-known anticancer drug, cisplatin. The interaction of the compounds with calf thymus DNA (CT-DNA) and human serum albumin (HSA) were assayed by a series of experimental techniques including electronic absorption, fluorescence, viscometry, gel electrophoresis, and FT-IR. The results indicated that the two complexes have interesting binding propensities toward CT-DNA as well as HSA and the binding affinity of (1) is more than (2). The fluorescence data indicated that both complexes strongly quench the fluorescence of ethidium bromide–DNA system as well as the intrinsic fluorescence of HSA via static quenching procedures. The thermodynamic parameters (ΔH°, ΔS°, and ΔG°) calculated from the fluorescence studies showed that hydrogen bonds and van der Waals interactions play a major role in the binding of the complexes to DNA and HSA. We suggest that both of the Pd(II) complexes exhibit the groove binding mode with CT-DNA and interact with the main binding pocket of HSA.

Communicated by Ramaswamy H. Sarma     

Mutant regulatory subunit of 3'',5''-cAMP-dependent protein kinase of yeast Saccharomyces cerevisiae

Summary Four mutants with amino acid substitution(s) at or near the putative phosphorylation site (Arg¹⁴² Arg¹⁴³ Thr¹⁴⁴ Ser¹⁴⁵) of the regulatory subunit of cAMP-dependent protein kinase were obtained by site-directed mutagenesis. Three mutants, BCY1 ^{Ala 145} (Ser¹⁴⁵ to Ala), BCY1 ^{His 143} (Arg¹⁴³ to His) and BCY1 ^{Asn 144, Ala 145} (Thr¹⁴⁴ to Asn and Ser¹⁴⁵ to Ala) complemented a bcy1 mutant, whereas BCY1 ^{Gly 143} (Arg¹⁴³ to Gly) did not. In addition, mutant, BCY1 ^{Asn 144, Ala 145} exhibited a dominant coldsensitive phenotype, which can be most easily explained by the functional alteration of the regulatory subunit of cAMP-dependent protein kinase by the mutations. Analyses of these mutant genes revealed that phosphorylation of the regulatory subunit is not a prerequisite for the regulation of the cAMP-dependent protein kinase activity in responding to the cAMP level.     

Effect of amino acids present at the carboxyl end of succinimidyl residue on the rate constants for succinimidyl hydrolysis in small peptides

Structural alterations of aspartyl and asparaginyl residues in various proteins can lead to their malfunction, which may result in severe health disorders. The formation and hydrolysis of succinimidyl intermediates are crucial in specific protein modifications. Nonetheless, only few studies investigating the hydrolysis of succinimidyl intermediates have been published. In this study, we established a method to prepare peptides bearing succinimidyl residues using recombinant protein l-isoaspartyl methyltransferase and ultrafiltration units. Using succinimidyl peptides, we examined the effect of amino acid residues on succinimidyl hydrolysis at the carboxyl end of succinimidyl residues and determined the rate constant of hydrolysis for each peptide. The rate constant of succinimidyl hydrolysis in the peptide bearing a Ser residue at the carboxyl side (0.50 ± 0.02 /h) was 3.0 times higher than that for the peptide bearing an Ala residue (0.17 ± 0.01 /h), whereas it was just 1.2 times higher for the peptide bearing a Gly residue (0.20 ± 0.01 /h). The rate constant of succinimidyl formation in the peptide bearing a Ser residue [(2.44 ± 0.11) × 10⁻³ /d] was only 1.2 times higher than that for the peptide bearing an Ala residue ([1.87 ± 0.09) × 10⁻³ /d], whereas 5.5 times higher for the peptide bearing a Gly residue [(10.2 ± 0.2) × 10⁻³ /d]. These results show that the Gly and Ser residues at the carboxyl end of the succinimidyl residue have opposing roles in succinimidyl formation and hydrolysis. Catalysis of Ser residue's hydroxyl group plays a crucial role in succinimidyl hydrolysis.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Mutation strategies for obtaining chitooligosaccharides with longer chains by transglycosylation reaction of family GH18 chitinase

Enhancing the transglycosylation (TG) activity of glycoside hydrolases does not always result in the production of oligosaccharides with longer chains, because the TG products are often decomposed into shorter oligosaccharides. Here, we investigated the mutation strategies for obtaining chitooligosaccharides with longer chains by means of TG reaction catalyzed by family GH18 chitinase A from Vibrio harveyi (VhChiA). HPLC analysis of the TG products from incubation of chitooligosaccharide substrates, GlcNAc_n, with several mutant VhChiAs suggested that mutant W570G (mutation of Trp570 to Gly) and mutant D392N (mutation of Asp392 to Asn) significantly enhanced TG activity, but the TG products were immediately hydrolyzed into shorter GlcNAc_n. On the other hand, the TG products obtained from mutants D313A and D313N (mutations of Asp313 to Ala and Asn, respectively) were not further hydrolyzed, leading to the accumulation of oligosaccharides with longer chains. The data obtained from the mutant VhChiAs suggested that mutations of Asp313, the middle aspartic acid residue of the DxDxE catalytic motif, to Ala and Asn are most effective for obtaining chitooligosaccharides with longer chains.     

Conformation of the central sequence of angiotensin II and analogs

We describe the synthesis and the conformational analysis by ir, CD, and proton-nmr spectroscopy of four model peptides of the type N-Ac-Tyr-X-His-NH₂ with X = Val, Leu, Ala, Gly. These peptides represent the central sequence of the hormone angiotensin II and its position-5 analogs. We studied their conformational behavior in aqueous solution during pH titration and in organic solvents. For specific purposes of spectral analysis (ir band assignment, proton-nmr signal assignment, heteronuclear vicinal coupling constants), we synthesized three isotopically enriched homologs of the mother sequence, i.e., N-Ac-(¹⁵N-Tyr)-Val-His-NH₂, N-Ac-(¹³C, ²H, Tyr)-Val-His-NH₂, and N-Ac-Tyr-(¹³C, ²H, Val)-His-NH₂. Results are summarized as follows: the tyrosine and the histidine side chains influence each other through space; this mutual influence is modulated by the nature of the side chain in position X and decreases in going from X?Val to X?Gly as a consequence of two simultaneous events, changes in the side-chain rotamer distribution and changes in the φ and ψ angles of residue X. The decrease in the bulkiness of the side-chain X (Val → Gly) leads to increased flexibility of the peptide backbone at this site, which is also reflected in the apparent ratio of C₅, C₇, and intermediate conformations present in equilibrium. The three spectroscopic techniques, in addition to the results of chymotryptic degradation experiments, show a high level of agreement, and all reflect the dynamic conformation of these peptides in a different manner.     

Mutations that modify or exclude binding of the QA ubiquinone and carotenoid in the reaction center from Rhodobacter sphaeroides

Three single-site mutations have been introduced at positions close to the Q_A ubiquinone in the reaction centre from Rhodobacter sphaeroides. Two of these mutations, Ala M260 to Trp and Ala M248 to Trp, result in a reaction centre that does not support photosynthetic growth of the bacterium, and in which electron transfer to the Q_A ubiquinone is abolished. In the reaction centre with an Ala to Trp mutation at the M260 residue, electron transfer from the primary donor to the acceptor bacteriopheophytin is not affected by the mutation, but electron transfer from the acceptor bacteriopheophytin to Q_A is not observed. The most likely basis for these effects is that the mutation produces a structural change that excludes binding of the Q_A ubiquinone. A third mutation, Leu M215 to Trp, produces a reaction centre that has an impaired capacity for supporting photosynthetic growth. The mutation changes the nature of ubiquinone binding at the Q_A site, and renders the site sensitive to quinone site inhibitors such as o- phenanthroline. Adopting a similar approach, in which a small residue located close to a cofactor is changed to a more bulky residue, we show that the reaction centre can be rendered carotenoid-less by the mutation Gly M71 to Leu.     

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.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

Frequent Missense and Insertion/Deletion Polymorphisms in the Ovine Shadoo Gene Parallel Species-Specific Variation in PrP

Background

The cellular prion protein PrP^C is encoded by the Prnp gene. This protein is expressed in the central nervous system (CNS) and serves as a precursor to the misfolded PrP^Sc isoform in prion diseases. The prototype prion disease is scrapie in sheep, and whereas Prnp exhibits common missense polymorphisms for V136A, R154H and Q171R in ovine populations, genetic variation in mouse Prnp is limited. Recently the CNS glycoprotein Shadoo (Sho) has been shown to resemble PrP^C both in a central hydrophobic domain and in activity in a toxicity assay performed in cerebellar neurons. Sho protein levels are reduced in prion infections in rodents. Prompted by these properties of the Sho protein we investigated the extent of natural variation in SPRN.

Principal Findings

Paralleling the case for ovine versus human and murine PRNP, we failed to detect significant coding polymorphisms that alter the mature Sho protein in a sample of neurologically normal humans, or in diverse strains of mice. However, ovine SPRN exhibited 4 missense mutations and expansion/contraction in a series of 5 tandem Ala/Gly-containing repeats R1-R5 encoding Sho''s hydrophobic domain. A Val71Ala polymorphism and polymorphic expansion of wt 67(Ala)₃Gly70 to 67(Ala)₅Gly72 reached frequencies of 20%, with other alleles including Δ67–70 and a 67(Ala)₆Gly73 expansion. Sheep V71, A71, Δ67–70 and 67(Ala)₆Gly73 SPRN alleles encoded proteins with similar stability and posttranslational processing in transfected neuroblastoma cells.

Significance

Frequent coding polymorphisms are a hallmark of the sheep PRNP gene and our data indicate a similar situation applies to ovine SPRN. Whether a common selection pressure balances diversity at both loci remains to be established.     

Purification and Characterization of a Co(II)-Sensitive α-Mannosidase from Ginkgo biloba Seeds

An α-mannosidase was purified from developing Ginkgo biloba seeds to apparently homogeneity. The molecular weight of the purified α-mannosidase was estimated to be 120 kDa by SDS–PAGE in the presence of 2-mercaptoethanol, and 340 kDa by gel filtration, indicating that Ginkgo α-mannosidase may function in oligomeric structures in the plant cell. The N-terminal amino acid sequence of the purified enzyme was Ala–Phe–Met–Lys–Tyr–X–Thr–Thr–Gly–Gly–Pro–Val–Ala–Gly–Lys–Ile–Asn–Val–His–Leu–. The α-mannosidase activity for Man₅GlcNAc₁ was enhanced by the addition of Co²⁺, but the addition of Zn²⁺, Ca²⁺, or EDTA did not show any significant effect. In the presence of cobalt ions, the hydrolysis rate for pyridylaminated Man₆GlcNAc₁ was significantly faster than that for pyridylaminated Man₆GlcNAc₂, suggesting the possibility that this enzyme is involved in the degradation of free N-glycans occurring in developing plant cells (Kimura, Y., and Matsuo, S., J. Biochem., 127, 1013–1019 (2000)). To our knowledge, this is the first report showing that plant cells contain an α-mannosidase, which is activated by Co²⁺ and prefers the oligomannose type free N-glycans bearing only one GlcNAc residue as substrate.     

Analogues of deltorphin I containing conformationally restricted amino acids in position 2: structure and opioid activity

New analogues of deltorphin I (DT I, Tyr‐d ‐Ala‐Phe‐Asp‐Val‐Val‐Gly‐NH₂), with the d ‐Ala residue in position 2 replaced by α‐methyl‐β‐azido(amino, 1‐pyrrolidinyl, 1‐piperidinyl or 4‐morpholinyl)alanine, were synthesized by a combination of solid‐phase and solution methods. All ten new analogues were tested for receptor affinity and selectivity to μ‐ and δ‐opioid receptors. The affinity of analogues containing (R) or (S)‐α‐methyl‐β‐azidoalanine in position 2 to δ‐receptors strongly depended on the chirality of the α,α‐disubstituted residue. Peptide II , containing (S)‐α‐methyl‐β‐azidoalanine in position 2, displayed excellent δ‐receptor selectivity with its δ‐receptor affinity being only three times lower than that of DT I. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.