Substance P in Solution: Trans-to-Cis Configurational Changes of Penultimate Prolines Initiate Non-enzymatic Peptide Bond Cleavages

Abstract

We report ion mobility spectrometry and mass spectrometry studies of the non-enzymatic step-bystep degradation of substance P (subP), an eleven-residue neuropeptide, with the sequence Arg 1-Pro 2-Lys 3-Pro 4-Gln 5-Gln 6-Phe 7-Phe 8-Gly 9-Leu 10-Met 11-NH 2 , in ethanol. At elevated solution temperatures (55 to 75 °C) several reactions are observed, including: a protonation event, i.e., [subP+2H] 2+ + H + → [subP+3H] 3+ , that appears to be regulated by a configurational change; and, two sequential bond cleavages (the Pro 2-Lys 3 peptide bond is cleaved to form the smaller nonapeptide Lys 3-Met 11-NH 2 [subP (3-11) ], and subsequently, subP (3-11) is cleaved at the Pro 4-Gln 5 peptide bond to yield the heptapeptide Gln 5-Met 11-NH 2 [subP (5-11) ]). Each of the product peptides [subP (3-11) and subP (5-11) ] is accompanied by a complementary diketopiperazine (DKP): cyclo-Arg 1-Pro 2 (cRP) for the first cleavage, and cyclo-Lys 3-Pro 4 (cKP) for the second. Insight about the mechanism of degradation is obtained by comparing kinetics calculations of trial model mechanisms with experimental data. The best model of our experimental data indicates that the initial cleavage of subP is regulated by a conformational change, likely a trans→cis isomerization of the Arg 1-Pro 2 peptide bond. The subP (3-11) product has a long lifetime (t ½ ~ 30 hrs at 55 °C) and appears to transition through several structural intermediates prior to dissociation, suggesting that subP (3-11) is initially formed with a Lys 3-trans-Pro 4 peptide bond configuration and that slow trans→cis isomerization regulates the second bond cleavage event as well. From these data and our model mechanisms, we obtain transition state thermochemistry ranging from ΔH ‡ = 41 to 85 kJ•mol −1 and ΔS ‡ = −43 to −157 J•mol −1 •K −1 for each step in the reaction.

Melting of Hemoglobin in Native Solutions as measured by IMS-MS

“…Recent work from our group and others has employed variable-temperature electrospray ionization (vT-ESI) with ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques to characterize the thermal stability of peptides, − proteins, − and protein complexes. − These studies provide evidence for populations of multiple distinct structures that vary with temperature. The combination of IMS (which provides information about conformer shape) with MS (which determines a protein’s charge state under different conditions) is especially well suited for determining the stoichiometry of protein complexes, − identifying post-translational modifications, − characterizing bound ligands, − as well as determining thermochemistry and activation energies associated with specific types of structural transitions. , Herein, we build upon earlier vT-ESI-IMS-MS work to examine the species involved in thermal denaturation of human hemoglobin A, a heterotetrameric complex.…”

Proline Behavior in Model Prebiotic Peptides Formed by Wet–Dry Cycling

“…Recent studies have shown a propensity for N-terminal peptide truncation and DKP formation with cis-Pro at the second position. 68,69 Therefore, it is reasonable to conclude that cis-Pro conformations did induce cyclization in our mixtures, albeit to a minor degree. It should be noted that the relative abundances of both sets of y 2 ions in Figure 5C,D reflect gas-phase ion stabilities and not solution-phase populations of hydroxy acids versus amino acids at N-termini.…”

A stochastic dynamic mass spectrometric diffusion method and its application to 3D structural analysis of the analytes

“…In a analogy with our methodology, the latter approach implements molecular dynamic simulations, in order to distinguish between most preferred conformations of the analyte with respect to the minimum of the potential energy. However, through the whole text of the paper we have underlined that only an assessment of the energy of the molecular systems is unable to distinguish precisely between different conformations (Conant et al, 2019). As mentioned before, the employment of D SD parameter, due to its great sensitivity, enables us to determine exactly the 3D molecular and electronic molecular structures based on soft-ionization MS experiment.…”

“…However, as can be seen from the example presented in the preceding section, the energy difference in the molecular conformations can be significantly lower, which prevents an accurate determination of the exact multidimensional molecular structure, because the aforementioned CCS does not correspond to the real CCS. The molecular CCS reflects average values of the geometry orientation of the analyte within the whole time of measurement (May, Morris & McLean, 2017;El-Baba et al, 2018;Conant et al, 2019). However, as our research, so far, has shown, the molecular conformation and the electronic interactions can be perturbed due to solute-continuum interactions under the MS experiment.…”