Glu Side Chains Research Articles

Crystal structure of the α‐helical undecapeptide Boc‐L‐Ala‐Aib‐Ala‐Aib‐Ala‐Glu(OBzl)‐Ala‐Aib‐Ala‐Aib‐Ala‐OMe

AbstractThe x‐ray structure of Boc‐L‐Ala‐Aib‐Ala‐Aib‐Ala‐Glu(OBzl)‐Ala‐Aib‐Ala‐Aib‐Ala‐OMe(I) represents the first α‐helix determined by direct methods. This undecapeptide is a model of the N‐terminus of alamethicin, and it exhibits voltage‐dependent pores in bilayer membranes at a higher voltage and concentration than alamethicin. The molecule crystallizes in the monoclinic space group P21 with a = 10.602(1), b = 23.884(3), c = 13.622(1) Å, β = 95.61(6)°, and Z = 2. It adopts a right‐handed α‐helical conformation in the solid state with intramolecular 5 → 1 hydrogen bonds. An additional intramolecular hydrogen bond is bifurcated, forming a stronger 4 → 1 interaction (i.e., a β‐turn III) and a weaker 5 → 1 interaction, thus prolonging the α‐helical part up to 9 residues. The α‐helix radius of 2.1 Å, the height per residue (distance Ni … Ni + 4) of 1.53 Å, the resulting length of the α‐helical part of 13.8 Å (9 residues) resp. 15.3 Å (10 residues), the van der Waals radius (4.7 Å), and the minimal diameter of pores formed by aggregation of 3–10 α‐helices were calculated omitting the Glu(OBzl) side chain. In the crystal, the α‐helices are linked head to tail via two hydrogen bridges forming continuous chains. Adjacent helices are oriented in antiparallel with their helix axes and have only van der Waals contacts.

Synthesis of partial sequences of ribosomal proteins and their derivatization with acryloyl residues

Peptide sequences B‐X‐B (B = Arg and/or Lys; X = Glu or Asp) are of considerable interest because of their possible interactions with ribosomal RNA. The syntheses of various protected peptides with the sequence Ala‐B‐X‐B‐Ala (X = Glu or Ala) and [Arg]n‐Pro (n = 1–3) are described. They are carried out in solution according to the conventional peptide synthesis method. The carbobenzoxy group is used for Nα‐protection and the methyl group for the protection of the terminal carboxyl group. The side chains of Lys and Glu are respectively blocked with the tert.‐butyloxycarbonyl and the tertiary butyl group and the guanidinic function of arginine with the NO2‐group. The intermediate peptides are purified either by extraction or by size exclusion chromatography. A specially adapted strategy of peptide synthesis allows removal of the amino terminal Cbo‐group at the end of the synthesis and introduction of an acryloyl group. By radical copolymerization with cross‐linking agents these acryloyl derivatives can be transferred into insoluble peptide gels suitable for affinity chromatography and for investigating peptide‐oligonucleotide interactions. The isolation of the unprotected peptides Arg‐Arg‐Arg‐Pro, Ala‐Arg‐Glu‐Arg‐Ala, Ala‐Arg‐Glu‐Lys‐Ala, Ala‐Arg‐Ala‐Lys‐Ala, Ala‐Lys‐Glu‐Lys‐Ala and their characterization using amino acid analysis, electrophoresis, and FAB‐mass spectrometry is also reported.

Conformations of cyclic peptide/calcium complexes in solution.

AbstractSynthetic cyclic octapeptides of general structure cyclo[Glu(γOBzl)‐Sar‐Gly‐(N‐R)Gly]2 (R = n‐hexyl and cyclohexyl) transport calcium ions selectively across organic phases and phospholipid membranes. We have now used proton nmr spectroscopy (360 MHz) to study the solution conformation(s) of their calcium complexes. When Ca(ClO4)2 was added to solutions of these peptides in CDCl3, nmr spectra of the resulting calcium complexes were characteristic of a single C2‐symmetric conformer. From a Karplus‐Bystrov analysis of vicinal coupling constants in both the peptide backbone and Glu side chain (treated as an ABCC′MX spin system), in conjuction with model‐building studies, a structure was proposed in which the calcium ion is bound in an octahedral‐type complex by the four (coplanar) carbonyl groups of the (all‐trans) Glu‐Sar and Gly‐(N‐R)Gly peptide bonds. Occurrence of preferred rotamers about Glu side chain Cα–Cβ bonds indicated that restricted rotation in peptide side chains arises upon calcium binding.

High‐resolution study of the helix–coil transition of poly(L‐glutamic acid): Spectroscopic data correlation and the discovery of a new transition

AbstractThis article reports on both (1) the precision and capability of a computerized multidimensional spectrophotometric system recently developed in our laboratory and (2) the high‐resolution study of the helix–coil transition of poly(L‐glutamic acid)[poly(Glu)], especially with regard to the discovery of an overlooked transition which is attributable to order–disorder rearrangement of the poly(Glu) side chain in the α‐helical conformation. This study was made possible by the high performance of the system used. The simultaneous and continuous measurement of the circular dichroism, the absorbance and light‐scattering intensity, and the pH titration curve of poly(Glu) in aqueous salt solution was carried out under continuous scanning of pH ranging from 8 to 2. Besides the well‐known random coil to α‐helix transition that occurs at about pH 5.5, a highly cooperative transition, which is indicated as a small but definite step in several spectral dimensions, is observed for the first time at pH 4.3. The transition is ascribed to an order–disorder conversion of the side chain on the α‐helix backbone.

1H‐nmr parameters of the common amino acid residues measured in aqueous solutions of the linear tetrapeptides H‐Gly‐Gly‐X‐L‐Ala‐OH

AbstractThe 1H‐nmr chemical shifts and the spin–spin coupling constants of the common amino acid residues were measured in solutions of the linear tetrapeptides H‐Gly‐Gly‐X‐L‐Ala‐OH in D2O and H2O, the influence of X on the nmr parameters of the neighboring residues Gly 2 and Ala 4 was investigated. The titration parameters for the side chains of Asp, Glu, Lys, Tyr, and His were determined. The pKa values obtained in D2O, with the use of pH‐meter readings with a combination glass electrode uncorrected for istope effects, were 0.06 pH units higher in the acidic range and 0.10 pH units higher in the basic range than the corresponding pKa values in H2O. This suggests that the present data are suitable “random‐coil” 1H‐nmr parameters for conformational studies of polypeptide chains in D2O and H2O solutions.

Piezoelectric relaxations in homopolymers and copolymers of γ-Benzyl-L-glutamate and L-Leueine

Abstract The temperature dependence of the piezoelectric stress-constant, the piezoelectric strain-constant, and the Young's elastic constant is simultaneously determined for homopolymers and copolymers of γ-benzyl-L-glutamate [Glu(OBz)] and L-leucine (Leu). With the rise of temperature, the piezoelectric constants first increase due to the elastic relaxation in the Leu side chains (about −150°C) and the Glu(OBz) side chains (about −10°C), and then decrease due to the dielectric relaxation in the Glu(OBz) side chains. Higher piezoelectric constants are observed for the copolymers at low temperatures. This suggests that the shear-induced internal strain of the peptide dipoles is enhanced by heterogenity in the structure of side chains.

Specific and irreversible inhibition of lysozyme by 2',3'-epoxypropyl beta-glycosides of N-acetyl-D-glucosamine oligomers.

THE structure and properties of hen's egg white lysozyme and of the lysozyme–inhibitor complexes are known in detail1,2, but there is no direct evidence as to which amino-acid residues are involved in the bond breaking step. Specific chemical modification of the active site residues is an obvious approach to the problem, arid we describe here an attempt to design specific inhibitors of lysozyme, using the affinity labelling principle3,4. Because the side chains of amino-acid residues Glu 35 and Asp 52 have been proposed as the catalytically active groups in lysozyme2, we have prepared potential lysozyme inhibitors which can react with carboxyl functions, and have investigated their effect on the enzyme.

Binding of p-nitrothiophenol to myosin a

The p-nitrothiophenol-myosin A compound was produced by reaction of p-nitrothiophenol with myosin A in the presence of Mg 2+ and ATP, and was isolated by gel filtration through a Sephadex column. The change in absorption spectrum by the binding of p-nitrothiophenol to myosin A was similar to that of the formation of a thiolester bond between p-nitrothiophenol and succinic anhydride. The p-nitrothiophenol-myosin A compound was stable at acidic and neutral pH, but was unstable at alkaline pH. The stability was not affected by replacement of air with N 2 gas. The p-nitrothiophenol bound to myosin A was easily displaced by cysteine and H 2S. p-Nitrothiophenyl-peptides were isolated with 24% recovery after proteolysis and chromatography on talc, since the proteolysis did not accelerate the liberation of p-nitrothiophenol. The hydromate was formed quantitatively by the addition of NH 2OH to p-nitrothiophenyl-peptides. The amino acid composition of the p-nitrothiophenyl-peptides was determined, and Arg, Asp, Ser, Glu, Ileu and Leu were found as amino acid residues whose mole ratios to p-nitrothiophenol were nearly equal to or higher than 1. These results establish the covalent binding of p-nitrothiophenol to myosin A in the presence of Mg 2+ and ATP, and strongly suggest the binding of p-nitrothiophenol on a carboxyl side-chain of Glu or Asp in myosin A as an acylthiol bond. Effects of divalent cations, pH and modifiers of the ATPase activity of the p-nitrothiophenyl-myosin A were also investigated. The pH-activity curve of the p-nitrothiophenyl-myosin A was similar to those of the p-chloromercuribenzoate-myosin A compound and the trinitrophenyl-myosin A, and showed no depression at neutral pH. The ATPase (ATP phosphohydrolase, EC 3.6.1.3) of the p-nitrothiophenyl-myosin A was activated by p-chloromercuribenzoate or EDTA, whereas the ATPase of the trinitrophenyl-myosin A was not activated. Actomyosin reconstituted from the p-nitrothiophenyl-myosin A showed ATPase activity of the typical actomyosin type.

Amino acid composition and N-terminal amino acid sequence of porcine secretin

A novel bioactive form of neurotensin post-translationally modified at a Glu residue was isolated from porcine intestine. Purification of the peptide was guided by detection of intracellular Ca2+ release in SK-N-SH neuroblastoma cells. Using high resolution accurate mass analysis on an ion trap Fourier transform mass spectrometer, the post-translational modification was identified as arginine linked to the γ-carboxyl of Glu via an isopeptide bond, and we named the newly identified peptide “arginylated neurotensin” (R-NT, N-(neurotensin-C5–4-yl)arginine). Although arginylation is a known modification of N-terminal amino groups in proteins, its presence at a Glu side chain is unique. The finding places neurotensin among the few physiologically active peptides that occur both in post-translationally modified and unmodified forms. Pharmacologically, we characterized R-NT for its ligand activity on three known neurotensin receptors, NTR1, -2, and -3, and found that R-NT has similar pharmacological properties to those of neurotensin, however, with a slightly higher affinity to all three receptors. We expressed the intracellular receptor NTR3 as a soluble protein secreted into the cell culture medium, which allowed characterization of its R-NT and neurotensin binding properties. The creation of soluble NTR3 also provides a potential tool for neutralizing neurotensin action in vivo and in vitro. We have shown that SK-N-SH neuroblastoma cells express NTR1 and NTR3 but not NTR2, suggesting that the Ca2+ mobilization elicited by R-NT is via NTR1.