N-Terminal Decarboxylation as a Probe for Intramolecular Contact Formation in γ-Glu-(Pro)_n-Met Peptides

Abstract

The kinetics of intramolecular-contact formation between remote functional groups in peptides with restricted conformational flexibility were examined using designed peptides with variable-length proline bridges. As probes for this motion, free radicals were produced using the • OH-induced oxidation at the C-terminal methionine residue of γ-Glu-(Pro) n -Met peptides ( n = 0–3). The progress of the radicals’ motion along the proline bridges was monitored as the radicals underwent reactions along the peptides’ backbones. Of particular interest was the reaction between the sulfur atom located in the side chain of the oxidized Met residue and the unprotonated amino group of the glutamic acid moiety. Interactions between them were probed by the radiation-chemical yields (expressed as G values) of the formation of C-centered, α-aminoalkyl radicals (αN) on the Glu residue. These radicals were monitored directly or via their reaction with p -nitroacetophenone (PNAP) to generate the optically detected PNAP •– radical anions. The yields of these αN radicals were found to be linearly dependent on the number of Pro residues. A constant decrease by 0.09 μM J –1 per spacing Pro residue of the radiation-chemical yields of G (αN) was observed. Previous reports support the conclusion that the αN radicals in these cases would have to result from (S∴N) + -bonded cyclic radical cations that arose as a result from direct contact between the ends of the peptides. Furthermore, by analogy with the rate constants for the formation of intramolecularly (S∴S) + -bonded radical cations in Met-(Pro) n -Met peptides ( 27513096 J. Phys. Chem. B 2016 120 9732 ), the rate constants for the formation of intramolecularly (S∴N) + -bonded radical cations are activated to the same extent for all of the γ-Glu-(Pro) n -Met peptides. Thus, the continuous decrease of G (αN) with the number of Pro residues (from 0 to 3) suggests that the formation of a contact between the S-atom in the C-terminal Met residue and the N-atom of a deprotonated N-terminal amino group of Glu is controlled in peptides with 0 to 3 Pro residues by the relative diffusion of the S •+ and unoxidized N-atom. The overall rate constants of cyclization to form the (S∴N)-bonded radical cations were estimated to be 3.8 × 10 6 , 1.8 × 10 6 , and 8.1 × 10 5 s –1 for peptides with n = 0, 1, and 2 Pro residues, respectively. If activation is the same for all of the peptides, then these rate constants are a direct indication for the end-to-end dynamics along the chain. ...

Oxidation of Designed Model Peptides Containing Methionine, Proline and Glutamic Acid Investigated by Tandem Mass Spectrometry and IRMPD Spectroscopy

“…Oxidation products þ16 Da and þ32 Da have been observed for both EM and EPM peptides as well as decarboxylation. The key transient a-aminoalkyl radicals (a N), formed after the loss of CO 2 , were found to linearly decreased for g-Glu-(Pro) n -Met peptides when n increased from 0 to 2 [14]. However, there is no difference in the yield of the final product related to decarboxylation.…”

“…Oxidation of peptides containing Methionine (M), Proline (P) and/or Glutamic acid (E) has been investigated by pulse radiolysis. Only free radicals coming from methionine were detected [14,15], suggesting that the other residues were protected from oxidation. Indeed the rate constants of oxidation by OH radicals with these residues are in favor of reactions with methionine mostly (k(OH þ Met) ¼ 8.5 Â 10 9 Mol À1 .L.s À1 ; k(OH þ Pro) ¼ 3.1 Â 10 8 Mol À1 .L.s À1 ; k(OH þ Glu) ¼ 2.3 Â 10 8 Mol À1 .L.s À1 ) [16].…”

Photo- and Radiation-Induced One-Electron Oxidation of Methionine in Various Structural Environments Studied by Time-Resolved Techniques

“…Th mechanism is in principle very similar to that presented for Ser-Met and Thr-Met excep for the fact that the open chain N-centered radical cation via establishment of the me someric form (H2N-CH-(COO • ))-peptide and subsequent α-fragmentation leads to α-am noalkyl type radicals and CO2 (Scheme 10). These radicals obtained from the N-terminal decarboxylation reaction can be easily probed via their reaction with p-nitroacetophenone (PNAP) leading to PNAP •− radical anion [80][81][82]. The following fact should be emphasized at this point: the radiation chemical yield of CO 2 measured for this dipeptide was not equal to the radiation chemical yield of α-aminoalkyl type radicals [80].…”

“…In other words, the peptide backbone in γ-Glu-Gly-Met-Gly is flexible enough to allow direct interaction between amine and sulfur moieties. The γ-Glu-(Pro) n -Met (n = 0-3) oligopeptides were chosen as oligopeptides with restricted conformational flexibility [82]. The observed continuous decrease of G(αN) with the number of Pro residues (from 0 to 3) indicates that formation of a contact between the S-atom in the C-terminal Met residue and the N-atom of a deprotonated N-terminal amino group of Glu is controlled by the relative diffusion of the S •+ and unoxidized N-atom.…”

Radiation- and Photo-Induced Oxidation Pathways of Methionine in Model Peptide Backbone under Anoxic Conditions

“…Herein we extend the identification and quantification of transient species by pulse radiolysis at pH 7 and of final products of γ-radiolysis by LC-MS and high-resolution MS/MS under anoxic conditions. The purpose of acetylation of the N -terminal amino group is to eliminate the fast intramolecular proton transfer from the amino group to the sulfuranyl moiety, which was suggested earlier as the main decay reaction pathway, while the esterification of the C -terminal carboxyl group eliminates its decarboxylation [ 8 , 9 , 17 , 21 , 22 ]. Therefore, the use of 1 allows to study the reaction of HO • with Met residue with no contribution of N - and C -terminal functional groups.…”