The vγ chain of fetal hemoglobin of the orangutan

Previous studies suggested that orangutans have nonallelic structural genes for their γ chains, because either a threonyl or an alanyl residue may occupy position 135. Further investigation has now detected that position 75 may have either an isoleucyl or a valyl residue. From available evidence, the isoleucyl and threonyl residues are in one chain and the valyl and alanyl in the other. Orangutans appear to be homozygous for the two types of nonallelic genes.     

Molecular orbital calculations on the conformation of polypeptides and proteins. V. Conformational energy maps and stereochemical rotational states of aliphatic residues

The quantum-mechanical calculations by the PCILO method on the conformation of amino acid residues of proteins have been extended to the valyl, leucyl, and isoleucyl residues. In distinction to the earlier “empirical” computations, the quantum-mechanical results indicate very similar energy contours for the stable conformations of the three residues. Their general outline is also similar to that of the alanyl residue, although reduced by about 25%. Contrary to the “empirical” computations, the present results predict that the region corresponding to the α-helix should be one of great stability for the three residues and in particular for the valyl residue. The quantum-mechanical results are in excellent agreement with the experimental conformations of the aliphatic residues in lysozyme and myoglobin. Their prediction as to the ready availability of the valyl residue in the α-helical conformation agrees moreover with Ptitsyn's statistical evaluation of the participation of this residue in the inner turns of the helical regions in six globular proteins. The maximum conformational space allowed for the aliphatic residues is somewhat smaller than that allowed for the aromatic ones, while the minimum conformational space (region of stability common to all the residues) is similar in both groups.     

Phosphorylation of synthetic peptide analogs of the phosphorylatable site of phosphorylase b with phosphorylase kinase.

A synthetic octapeptide of the phosphorylatable site of phosphorylase and its analogs were used to determine the specificity of nonactivated phosphorylase kinase. By substitution of each of six amino acid residues (lysine₁₁, glutamine₁₂, isoleucine₁₃, serine₁₄, valine₁₅, and arginine₁₆), it was found that these residues were all important in the enzyme recognition. Valine₁₅ was more important than isoleucine₁₃, when either valine₁₅ or isoleucine₁₃ was substituted by glutamic acid. A peptide containing two isoleucyl residues (surrounding serine₁₄) had a better phosphorylation rate than a peptide containing two valyl residues. A peptide with a threonine residue instead of serine could be phosphorylated but with a low reaction rate.     

Stereospecific assignments of 1H-nmr methyl lines and conformation of valyl residues in the lac repressor headpiece

Two-dimensional ¹H-nmr methods are described to obtain information on the sidechain conformation of valyl residues of the lac repressor headpiece and to assign the resonances of their methyl groups stereospecifically. The spin–spin coupling constants (J_αβ) between C^αand C^β protons are obtained from two-dimensional correlated spectroscopy experiments. Large values for J_αβ(10–12 Hz) corresponding to trans orientations for these protons (g⁺ conformation) are found for all valyl residues in α-helical segments. For these valyl residues, the distance between one methyl group (γ₁)and the valyl amide proton is much shorter than for the other methyl group, so that stereospecific resonance assignments follow from relative intensities of the corresponding cross peaks in a two-dimensional nuclear Overhauser enhancement spectrum. Thus, streospecific assignments could be made for the methyl groups of Val 9, 20, 23, and 38 (of a total of eight valyl residues).     

Isolation and properties of goat beta 2-microglobulin

Goat beta 2-microglobulin was isolated and purified from colostrum. Comparisons of the amino acid composition and amino-terminal sequence of the goat protein with the bovine and human homologues, indicates a high degree of similarity. Both goat and bovine beta 2-microglobulins differ slightly in composition from the human molecule, most notably in threonine and proline values. For the first 32 residues, bovine and goat differ only at two positions, one of which is a valyl/isoleucyl substitution consistent with the amino acid compositions. The equivalent goat/human sequence comparison shows seven differences. Immunological studies, using the ELISA method, also confirm the close relatedness of goat and bovine beta 2-microglobulin and their more distant relatedness to the human homologue.     

A comparative study of essential arginine residues in Gramicidin S synthetase 2 and isoleucyl tRNA synthetase

In gramicidin S synthetase 2 (GS 2) from Bacillus brevis, L-proline, L-valine, L-ornithine, and L-leucine activations to aminoacyl adenylates are progressively inhibited by phenylglyoxal. The inactivation of GS 2 obeys pseudo-first-order kinetics. ATP completely prevents inactivation of GS 2 by phenylglyoxal, whereas amino acids only partially prevent it. In the presence of ATP, four arginine residues per mol of GS 2 are protected from modification by phenylglyoxal as determined by amino acid analysis and the incorporation of [7-14C]phenylgloxal into the enzyme protein, indicating that a single arginine residue is necessary for each amino acid activation. In isoleucyl tRNA synthetase from Escherichia coli, phenylglyoxal inhibits activation of L-isoleucine to isoleucyl adenylate. ATP completely prevents inactivation, although isoleucine only partially prevents it. One arginine residue of isoleucyl tRNA synthetase is protected by ATP from modification by phenylglyoxal, suggesting that a single arginine residue is essential for isoleucine activation. These results support the involvement of arginine residues in ATP binding with GS 2 or isoleucyl tRNA synthetase, and thus indicate that arginine residues of amino acid activating enzymes are essential for the formation of aminoacyl adenylates in both nonribosomal and ribosomal peptide biosynthesis.     

Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli. 2. Leucine resonance assignments by long-range coherence transfer

Sequence-specific assignments of the NH, C alpha H, and C beta H resonances in the NMR spectrum of the histidine-containing protein (HPr) from Escherichia coli are complete [Klevit, R. E., Drobny, G. P., & Waygood, E. B. (1986) Biochemistry (first paper of three in this issue)]. In addition, the C gamma H3 resonances of valyl, threonyl, and isoleucyl residues have been assigned by two-dimensional relayed coherence transfer (RELAY) experiments. In order to rigorously assign the resonances from longer side chains such as leucines, long-range transfer experiments have been applied to HPr. Coherence transfers via isotropic mixing within large spin systems were accomplished by multiple pulse trains applied during the mixing time of a two-dimensional experiment.     

Synthesis and activity of analogues of the isoleucyl tRNA synthetase inhibitor SB-203207

Twenty two analogues of SB-203207 have been prepared by total synthesis, and evaluated as inhibitors of a range of tRNA synthetases. Changes to the bicyclic core, removing either the terminal amino substituent or the sulfonyl group from the side chain, and altering either the carbon skeleton or stereochemistry of the isoleucine residue, decreases the potency of inhibition of isoleucyl tRNA synthetase. Substituting the isoleucine residue with other amino acids produces inhibitors of the corresponding synthetases. In particular, a methionine derivative is 50-100 times more potent against methionyl tRNA synthetase than against any of the corresponding isoleucyl, leucyl, valyl, alanyl and prolyl synthetases.     

Analogues of SB-203207 as inhibitors of tRNA synthetases

SB-203207 and 10 analogues have been prepared, by elaboration of altemicidin, and evaluated as inhibitors of isoleucyl, leucyl and valyl tRNA synthetases (IRS, LRS, and VRS, respectively). Substituting the isoleucine residue of SB-203207 with leucine and valine increased the potency of inhibition of LRS and VRS, respectively. The leucine derivative showed low level antibacterial activity, while several of the compounds inhibited IRS from Staphylococcus aureus WCUH29 more strongly than rat liver IRS.     

Synthesis of β-Peptide Standards for Use in Model Prebiotic Reactions

A one-pot method was developed for the preparation of a series of β-alanine standards of moderate size (2 to ≥12 residues) for studies concerning the prebiotic origins of peptides. The one-pot synthesis involved two sequential reactions: (1) dry-down self-condensation of β-alanine methyl ester, yielding β-alanine peptide methyl ester oligomers, and (2) subsequent hydrolysis of β-alanine peptide methyl ester oligomers, producing a series of β-alanine peptide standards. These standards were then spiked into a model prebiotic product mixture to confirm by HPLC the formation of β-alanine peptides under plausible reaction conditions. The simplicity of this approach suggests it can be used to prepare a variety of β-peptide standards for investigating differences between α- and β-peptides in the context of prebiotic chemistry.     

Peptide homologs, isosteres, and isomers: a general approach to structure-activity relationships

A general approach to study peptide structure is presented using three areas of ongoing research in our laboratories. The first involves the molecular basis for taste of peptide derivatives. We synthesized dipeptides based on L -aspartyl-α-aminocycloalkane carboxylic acid methyl ester. A homologous series of cycloalkane derivatives was studied. The cyclopropane, cyclobutane, and cyclopentane derivatives are sweet, the cyclohexane and cycloheptane peptides are bitter, and the cyclooctane homolog is tasteless. The related acyclic analog L -aspartyl-aminoisobutyric acid methyl ester is sweet, while the L -aspartyl diethyl glycine carboxylic acid methyl ester is tasteless. A model is presented to explain these experimental observations. The second area involves depsipeptides as isosteric replacements of α-hydroxy acids for amino acid residues in peptide chains. We have synthesized sequentially defined polydepsipeptides as model systems for polypeptides. A detailed analysis of the conformational order for these polydepsipeptides is presented. The third area involves partial retro–inverso peptide modifications of isomeric cyclic enkephalin analogs, which illustrate the relationship between the modification and biological activity. We are probing the intramolecular hydrogen-bonding features for these biologically active molecules. From such findings we are relating the structural and conformational preferences deduced from spectroscopy and molecular mechanics to biological activity.     

Development of aminoacyl tRNA synthetases in cultured Nicotiana tabacum cells

Changes in the activity of aminoacyl tRNA synthetases during growth of tobacco XD cells in suspension culture have been determined by the pyrophosphate exchange assay. Alanyl, arginyl, glutamyl, glutaminyl and seryl tRNA synthetases showed the lowest activity, whilst lysyl, histidyl, leucyl, isoleucyl, phenylalanyl threonyl and valyl tRNA synthetases were most active. Most synthetases, and total protein, increased to a maximum, at around 7 days, just before mid-exponential phase, and then fell.     

Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling

Stereospecific 1H and 13C NMR assignments were made for the two diastereotopic methyl groups of the 14 valyl and leucyl residues in the DNA-binding domain 1-69 of the 434 repressor. These results were obtained with a novel method, biosynthetically directed fractional 13C labeling, which should be quite widely applicable for peptides and proteins. The method is based on the use of a mixture of fully 13C-labeled and unlabeled glucose as the sole carbon source for the biosynthetic production of the protein studied, knowledge of the independently established stereoselectivity of the pathways for valine and leucine biosynthesis, and analysis of the distribution of 13C labels in the valyl and leucyl residues of the product by two-dimensional heteronuclear NMR correlation experiments. Experience gained with the present project and a previous application of the same principles with the cyclic polypeptide cyclosporin A provides a basis for the selection of the optimal NMR experiments to be used in conjunction with biosynthetic fractional 13C labeling of proteins and peptides.     

Partial characterization of a novel cathepsin L-like protease from Fasciola hepatica

A 30-kDa protease, purified previously from Fasciola hepatica, was sequenced and the first 15 N-terminal residues were found to be 100% homologous to a region in the protein Fcp1c, which was cloned and expressed from F. hepatica. This terminal region was also 53 and 54% identical to two other cathepsin L-like proteases isolated from the same source. The 30-kDa protease demonstrated a specificity different from humancathepsin L when assayed with novel peptidyl enediones of the type Z-Phe-Ala-CH&dbond;CH(2)-CO(2)R (where R = Me/Et/Bu(t)). The ethyl ester peptide was a more efficient inhibitor of the protease than the corresponding methyl ester. This is in contrast to bovine cathepsin B and human cathepsin L where both are more readily inhibited by the methyl, rather than the ethyl ester peptide. These differences in the inhibition of the novel parasite protease may allow it to be exploited as a chemotherapeutic target.     

Mutational separation of two pathways for editing by a class I tRNA synthetase

Aminoacyl tRNA synthetases (aaRSs) catalyze the first step in protein biosynthesis, establishing a connection between codons and amino acids. To maintain accuracy, aaRSs have evolved a second active site that eliminates noncognate amino acids. Isoleucyl tRNA synthetase edits valine by two tRNA(Ile)-dependent pathways: hydrolysis of valyl adenylate (Val-AMP, pretransfer editing) and hydrolysis of mischarged Val-tRNA(Ile) (posttransfer editing). Not understood is how a single editing site processes two distinct substrates--an adenylate and an aminoacyl tRNA ester. We report here distinct mutations within the center for editing that alter adenylate but not aminoacyl ester hydrolysis, and vice versa. These results are consistent with a molecular model that shows that the single editing active site contains two valyl binding pockets, one specific for each substrate.     

Aryl sulfotransferase IV from rat liver

A group of aryl sulfotransferases has been identified that catalyzes sulfate ester formation with simple phenols at an acidic pH and with several physiological metabolites at a more alkaline pH. One enzyme, aryl sulfotransferase IV, has been purified to homogeneity and found to be a protein of 61,000 daltons composed of two subunits of apparent equal size. Homogeneous preparations are active with simple phenols, organic hydroxylamines, and catecholamines as well as serotonin and its metabolites. The enzyme is also active with tyrosine methyl ester and with those peptide hormones e.g., cholecystokinin heptapeptide and some of the enkephalins, which have N-terminal tyrosine residues.     

Chain reversals in model peptides: studies of cystine-containing cyclic peptides. II. Effects of valyl residues and possible i-to-(i + 3) attractive ionic interactions on cyclization of [Cys1], [Cys6] hexapeptides

The synthesis of four N-acetyl N'-methylamide cystine-containing hexapeptides, CVPGVC, CGVVGC, CKPGEC, and CEPGKC, is described. These were used in disulfide-exchange reactions with the peptide CVPGGC as the formal oxidant. The relative propensities for peptide cyclization were thus deduced, and the tendency toward the formation of chain-reversal conformations was established quantitatively. An additional peptide, CVVVVC, was prepared but was never obtained as the cyclic monomer, demonstrating that the formation of chain-reversals in this peptide was of very low probability. Incorporation of pairs of valyl residues decreased the ease of cyclization, but it appeared that conformational flexibility in the cystine-containing hexapeptides may have compensated for substitutions which would have been expected to hinder the adoption of certain beta-turn conformations. The peptides containing ionic residues were cyclized more readily than expected, and this process was relatively insensitive to salt concentration. This observation is discussed with regard to the stabilization of beta-turns by i-to-(i + 3) ionic interactions in peptides and proteins. A method for blocking thiols was introduced as an improvement in the analysis of the equilibrium mixtures.     

An Electrophoretically Silent Polymorphism for the Beta Chains of Rabbit Hemoglobin and Associated Polyribosome Patterns

The beta chain of rabbit (Oryctolagus caniculus) hemoglobin has previously been reported to contain a single residue of isoleucine at beta(112). We have detected other rabbits with either zero isoleucyl residues or half a residue per beta chain. This character is polymorphic and inherited as a simple mendelian autosomal codominant.-Normally the modal number of ribosomes per polyribosome is 4 to 6 in reticulocyte lysates; but incubation of rabbit reticulocytes prior to lysis with L-o-methylthreonine (OMT), an isostere of isoleucine, leads to a bimodal distribution in lysates with 2-3 and 8-12 ribosomes as modes. This alteration has been attributed to ribosomal traffic jams caused by starvation for ile-tRNA at mRNA codons corresponding to the locations of isoleucyl residues at positions alpha(10), alpha(17), alpha(55) and beta(112). We have confirmed this interpretation by incubating OMT with reticulocytes from rabbits with integral, half integral and nil values for isoleucyl residues per beta chain to show that formation of the larger clusters of polyribosomes requires that beta(112) = ile.     

Studies on trypsin inhibitors. Part VIII. Synthesis of the protected octatriacontapeptide corresponding to the sequence 15-52 of porcine pancreatic secretory trypsin inhibitor II (Kazal)

The synthesis by fragment condensation of protected peptides corresponding to the amino acid sequences 15-35, 25-52 and 15-52 of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The Rudinger modification of the azide procedure was used in the fragment coupling steps. The tert-butyloxycarbonylheptapeptide hydrazide (sequence 22-28) was reacted with the heptapeptide methyl ester free base (sequence 29-35) and the resulting tert-butyloxycarbonyltetradecapeptide methyl ester after selective deprotection, coupled with the benzyloxycarbonylheptapeptide hydrazide (sequence 15-21) to give the protected peptide methyl ester corresponding to the 15-35 sequence which was then converted to the corresponding hydrazide. The synthesis of the 25-52 sequence was achieved by assembling the protected peptide hydrazide corresponding to the amino acid residues 25-35, with the C-terminal heptadecapeptide 36-52. The resulting protected octaeicosapeptide (sequence 25-52) was selectively deblocked with trifluoroacetic acid and acylated with the benzyloxycarbonyldecapeptide hydrazide 15-24 to give the desired octatriacontapeptide corresponding to sequence 15-52 of the inhibitor. An attempt to prepare the 15-52 sequence through the condensation of fragments corresponding to 15-35 and 36-52 sequences was unsuccessful. The identity and purity of the synthetized peptide derivatives wre established by elemental analysis (in some cases), amino acid analysis, optical rotation, and thin-layer chromatography in two solvent systems. The final products were also evaluated, after partial deprotection with anhydrous hydrogen fluoride or aqueous 90% trifluoroacetic acid, by paper electrophoresis at different pH values.     

Induction of beta-sheet structure in amyloidogenic peptides by neutralization of aspartate: a model for amyloid nucleation.

Amyloid fibril formation is widely accepted as a critical step in all types of amyloidosis. Amyloid fibrils derived from different amyloidogenic proteins share structural elements including beta-sheet secondary structure and similar tertiary structure. While some amyloidogenic proteins are rich in beta-sheet in their soluble form, others, like Alzheimer beta-amyloid peptide (Abeta) or serum amyloid A, must undergo significant structural transition to acquire a high beta-sheet content. We postulate that Abeta and other amyloidogenic proteins undergo a transition to beta-sheet as a result of aging-related chemical modifications of aspartyl residues to the form of succinimide or isoaspartyl methyl ester. We hypothesize that spontaneous cyclization of aspartate residues in amyloidogenic proteins can serve as a nucleation event in amyloidogenesis. To test this hypothesis, we synthesized a series of designed peptides having the sequence VTVKVXAVKVTV, where X represents aspartic acid or its derivatives. Studies using circular dichroism showed that neutralization of the aspartate residue through the formation of a methyl ester or an amide, or replacement of aspartate with glutamate led to an increased beta-sheet content at neutral and basic pH. A higher content of beta-sheet structure correlated with increased propensity for fibril formation and decreased solubility at neutral pH.