Cis-trans isomerizations of proline residues are key to bradykinin conformations

Abstract

A recent ion mobility-mass spectrometry (IM-MS) study of the nonapeptide bradykinin (BK, amino acid sequence Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)) found evidence for 10 populations of conformations that depend upon the solution composition [J. Am. Chem. Soc. 2011, 133, 13810]. Here, the role of the three proline residues (Pro(2), Pro(3), and Pro(7)) in establishing these conformations is investigated using a series of seven analogue peptides in which combinations of alanine residues are substituted for prolines. IM-MS distributions of the analogue peptides, when compared to the distribution for BK, indicate the multiple structures are associated with different combinations of cis and trans forms of the three proline residues. These data are used to assign the structures to different peptide populations that are observed under various solution conditions. The assignments also show the connectivity between structures when collisional activation is used to convert one state into another.

Figure 1

Collision cross section distributions of (a) [BK+3H]³⁺, (b) [BK-methyl+3H]³⁺, and (c) [acetyl-BK+3H]³⁺. The three most abundant features, labeled A, B, and C, appear analogous between BK and the BK-methyl. No [M+3H]³⁺ ions were observed for acetylated BK, as shown by the 1000× zoom (dashed line) of the baseline.

Figure 2

Collision cross section distributions for [M+3H]³⁺ ions of the three single Pro→Ala substituted BK analogue peptides (red traces) overlaid with the distribution of [BK+3H]³⁺ (dashed trace). For direct comparison of the analogues to BK, the cross section scales for the three analogue peptides were shifted according to amino acid size parameters from ref. 33 to account for the Pro→Ala substitution; see Experimental section for details. Intensities were normalized to peak maxima of BK for ease of visualization.

Figure 3

Comparison of collision cross section distributions of the three [M+3H]³⁺ double Pro→Ala substituted peptide analogues (red traces) with BK (dashed trace). Amino acid size parameters from ref. 33 were used to calibrate cross section scales of the analogues to BK. Intensities were normalized to peak maxima of BK for ease of visualization.

Figure 4

Cross section distribution of Pro^2,3,7→Ala BK [M+3H]³⁺ (RAAGFSAFR) (red) and [BK+3H]³⁺ (dashed). Amino acid size parameters from ref. 33 were used to calibrate cross section scales of the analogues to BK (see text for details). Intensities were normalized to peak maxima of BK to aid in comparison of the two distributions.