Glutamine Side Chains Research Articles

Thymine methyls and DNA-protein interactions.

Evidence is summarized showing that thymine methyls are as important in the recognition of specific sequences by proteins as are the more widely recognized hydrogen bonding sites of bases in the major groove (1). Strongest evidence has come from experiments using functional group mutagenesis (2) in which thymines in a specific recognition sequence (e.g., promoters, operators and restriction sites) are replaced by oligonucleotide synthesis with methyl-free uracil or cytosine and 5-methylcytosine. Such experiments have shown that thymine methyls can provide contact points via van der Waals interactions with amino acid side chains of specific DNA binding proteins. Actual contact between a thymine methyl and carbons of a glutamine side chain has been observed in a cocrystal of the phage 434 repressor and its operator by X-ray analysis. The issue of why thymine occurs in DNA is discussed in light of these findings.

Chemical mutations of the catalytic carboxyl groups in lysozyme to the corresponding amides.

In a two-step process, esterification and ammonolysis, Glu-35 and Asp-52 in lysozyme were amidated to glutamine and asparagine residues. Since the side chains of glutamine and asparagine are almost equal in size to those of glutamic acid and aspartic acid, these conversions would provide appropriate derivatives to elucidate the catalytic participations of these residues. The enzymatic activities of the resulting [Gln35]lysozyme and [Asn52]lysozyme were found to be less than 4% of that of native lysozyme in a pH range of 3.4-8.0. As these derivatives were inactive, we could determine the dissociation constants (Ks values) for the binding of beta-1,4-linked n-mer, a hexasaccharide of N-acetyl-D-glucosamine, to [Gln35]lysozyme and [Asn52] lysozyme. The values of Ks at pH 5.5 and 40 degrees C were 1.6 X 10(-5) M for [Gln35]lysozyme and 2.7 X 10(-5) M for [Asn52]lysozyme. These values are similar to that for native lysozyme. The results are direct proof for the involvements of Glu35 and Asp52 in the catalytic action of lysozyme. A method for ammonolysis of ester groups in proteins in liquid ammonia is described and will be useful for amidation of carboxyl groups of proteins.

Reactions of the amide side-chains of glutamine and asparagine in vivo

Reactions of the amide side-chains of glutamine and asparagine in vivo

Temperature and pH dependence of proton N.M.R. of glutamine in peptides

The proton n.m.r. spectra of the peptides Ala-Glu-Gly-Gln-Gly and Gly- Gln-Gly as measured in dimethyl sulfoxide over a range of temperatures yielded kinetic parameters for rotation about the carbon-nitrogen bonds of the glutamine side chains of ΔH‡ 71.6 kJ mol-1, ΔS‡ 2.1 J mol-1 K-1, and ΔH‡/70.6 kJ mol-1, ΔS‡ -0.5 J mol-1 K-1, respectively, at 298 K. On pH titration in dimethyl sulfoxide/water solutions, the chemical shifts of the cis protons of the glutamine amide remained constant whereas those of the trans protons were altered with a pK corresponding to that of the terminal glycine.

Nuclear magnetic resonance of labile protons in the basic pancreatic trypsin inhibitor

In the 1H nuclear magnetic resonance (n.m.r.) spectrum of the basic pancreatic trypsin inhibitor in 2H 20 at p 2H 4·6 and 36°C, resonances of 33 labile protons were identified. Additional labile protons were observed in H 2O. All the labile protons observed in 2H 20 come from amide groups of the polypeptide backbone and the side-chains of asparagine and glutamine. Individual assignments for 18 amide protons have been previously described (Dubs et al., 1979). Here, the backbone amide protons are characterized by their chemical shifts, the chemical shifts of the at-protons and, in so far as possible, of the side-chain protons, the spin-spin coupling constants with the α-protons, and the stability with respect to exchange with solvent protons. The spectral parameters are analyzed on the basis of the known locations of individual protons in the spatial structure of the protein. In H 2O, the hydrogen-bonded amide protons of the α-helix and the β-pleated sheet in the trypsin inhibitor gave well-resolved n.m.r. lines, even at extreme values of pH and temperature, showing that the conformation observed in single crystals is preserved over a pH range from at least 1·0 to 12·0 at 25°C, and at temperatures up to at least 90°C at neutral pH. The Appendix presents a Table with all the presently known individual proton resonance assignments in the trypsin inhibitor, including all the known spin systems with labile protons. This is the first in a series of three papers on n.m.r. studies of the amide protons in the basic pancreatic trypsin inhibitor.

Properties of metal complexes in the interphase of an oil continuous microemulsion. 2. Interaction of copper(II) with the side chains of lysine, glutamine, and methionine

Properties of metal complexes in the interphase of an oil continuous microemulsion. 2. Interaction of copper(II) with the side chains of lysine, glutamine, and methionine

Hydrogen-deuterium exchange of a primary amide as a model for asparagine and glutamine exchange in proteins

Hydrogen-deuterium exchange of the primary amide, isobutyramide, was investigated as a model for asparagine and glutamine-NH 2 exchange in a protein. A simple amide was chosen since the structures of several well-characterized proteins show most of these residues to be exposed to solvent. Isobutyramide-exchange data were obtained in 1:1 D 2O:dioxane solutions using a near-infrared method. The rate data were strictly pseudo-first order and yielded an average of 95% exchange of the primary amide hydrogens. In analogy with secondary amides, the pD dependence of the rate constants was characteristic of specific acid and base catalysis. In addition, analysis of the rate-pD profile for isobutyramide indicated a significant uncatalyzed exchange reaction. Temperature-dependence studies of the first-order rate constants at a fixed pD yielded an apparent activation energy of 19.3 kcal/mole. Predicted half-life times for the exposed primary amide hydrogens in proteins, based on these exchange parameters, indicate that asparagine and glutamine side chains generally would contribute to the overall rate data only below 15°C and then only for approximately 1 pD unit around the point of minimum reaction velocity.

Amino Acids and Peptides. XI. Phosphorus in Organic Synthesis. VI. Application of Diphenyl Phosphorazidate to the Synthesis of Peptides containing Various Functions

Application of diphenyl phosphorazidate (DPPA) to the synthesis of peptides revealed that the DPPA method is quite general because it can be applied to the formation of peptides containing various functional groups. No difficulties were encountered when the side chains of serine, threonine, valine, glutamine, asparagine, methionine, histidine, tryptophan, and pyroglutamic acid were present in the carboxyl component. Arginine and cysteine showed no trouble when their side chain functions were protected with the nitro and benzyl groups, respectively. Serine, tyrosine, and histidine could be used as the amino component without any protection of the side chains. Fragment coupling of N-benzyloxycarbonylleucylleucine with valylphenylalanine methyl ester hydrochloride by DPPA showed that no racemization occurred.

The Sites of Reaction of Wool with Formaldehyde

A method for the characterization of the products of the reaction between wool and formaldehyde is described. The method involves the use of a mild procedure to hydrolyze the formaldehyde-treated wool using digestion with proteolytic enzymes, and subsequent chromatographic separation of the resultant amino acids. Lysine and glutamine side chains have been identified as two of the sites of reaction.

The surface chemistry of wheat gluten I. Surface pressure measurements

Wheat gluten dispersed in a 0.1 M solution of hydrogen chloride in anhydrous chloroethanol was spread at the A W and the O W interface and the surface pressure (π) of the film measured as a function of the area ( A) over a wide range of pH (1–13) at the ionic strengths of 0.02 and 0.1. Some measurements were carried out at the interface between oil and 10% sodium salicylate or 24% urea. Gluten films were found to be more compressible and more stable than those of most other proteins. Pressures as high as 32 dynes/cm. ( A W ) and 36 dynes/cm. ( O W ) were obtained at an area of 0.2 m. 2/mg. while the minimum compressibility was 0.05 cm./dyne. The extrapolated area of close-packing was 1.33 m. 2/mg. at the A W and 1.54 m. 2/mg. at the O W interface. The area of minimum compressibility was 1.1 m. 2/mg. at both interfaces. At the O W interface variation in the pH of the substrate caused differences in the degree of expansion of the films at higher areas but all the π- A isotherms between pH 1 and 12 met at about the area of minimum compressibility. The area at constant pressure (1 dyne/cm.) as a function of pH gave a W-shaped curve showing minima at about pH 5.5 and 8.5 and a maximum at about pH 7.5. Variation in ionic strength from 0.02 to 0.1 had no effect on the minima; at the lower ionic strength the curve was flatter at the maximum and at the extreme ends. At pH 13 the π- A isotherm was quite different, indicating reduced cohesion in the film. The isotherm at the oil/aqueous salicylate interface was very similar to the pH 13 curve. At the oil/aqueous urea interface the isotherm was much expanded at the higher areas but met the control curve at about the area of minimum compressibility. It is concluded that spread gluten forms strongly bonded, coherent films, parts of which, when compressed below the area at which the films may be regarded as a close-packed monolayer, are displaced from the interface with relative ease and are folded up into loops. The unusually strong cohesion shown by these films may be due in part to hydrogen bonding between the numerous glutamine side chains, modified by electrostatic attraction. The rather high areas of close-packing and of minimum compressibility are probably due to the relatively high proline content of gluten.