Conformations of Prolyl−Peptide Bonds in the Bradykinin 1−5 Fragment in Solution and in the Gas Phase Liudmila Voronina, † Antoine Masson, †,∥ Michael Kamrath, † Franziska Schubert, ‡ David Clemmer, § Carsten Baldauf,* ,‡ and Thomas Rizzo* ,† † Laboratoire de Chimie Physique Molé culaire, E ́ cole Polytechnique Fe ́ de ́ rale de Lausanne, EPFL SB ISIC LCPM, Station 6, CH-1015 Lausanne, Switzerland ‡ Fritz-Haber-Institut der Max-Planck-Gesellschaft, D-14195 Berlin, Germany § Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States * S Supporting Information ABSTRACT: The dynamic nature of intrinsically disordered peptides makes them a challenge to characterize by solution-phase techniques. In order to gain insight into the relation between the disordered state and the environment, we explore the conformational space of the N- terminal 1−5 fragment of bradykinin (BK[1−5] 2+ ) in the gas phase by combining drift tube ion mobility, cold-ion spectroscopy, and first- principles simulations. The ion-mobility distribution of BK[1−5] 2+ consists of two well-separated peaks. We demonstrate that the conformations within the peak with larger cross-section are kinetically trapped, while the more compact peak contains low-energy structures. This is a result of cis−trans isomerization of the two prolyl-peptide bonds in BK[1−5] 2+ . Density-functional theory calculations reveal that the compact structures have two very different geometries with cis− trans and trans−cis backbone conformations. Using the experimental CCSs to guide the conformational search, we find that the kinetically trapped species have a trans−trans configuration. This is consistent with NMR measurements performed in a solution, which show that 82% of the molecules adopt a trans−trans configuration and behave as a random coil. 1. INTRODUCTION Information about the structure and dynamics of proteins and peptides is crucial for understanding their physiological function and hence essential for diagnostics and drug design. 1,2 X-ray crystallography and NMR spectroscopy can often successfully determine the structure of biomolecules when they adopt well-defined secondary structures. 3 However, in the case of intrinsically disordered peptides, NMR spectra often represent an average over an ensemble of di fferent conformations. Moreover, crystallization for X-ray measure- ments is often not possible due to the highly dynamic character of the disordered states. 4−6 Many questions thus remain open regarding the nature of intrinsically disordered peptides: Does their flexibility come from solution conditions? What are the main factors contributing to the rapid structural changes? How many well-defined conformers are converting among each other? How does recognition by receptors or membrane insertion happen from a structural perspective? 1,7,8 One of the most well-studied representatives of partially disordered peptides is the nonapeptide bradykinin (BK), which plays a regulatory role in the cardiovascular and nervous systems and is a key reporter molecule in inflammation and pain. 9 It was shown that in pure aqueous solution the entire peptide exhibits many conformational states rather than a single, well-defined secondary structure. 10 In an aprotic solvent or upon interaction with lipid vesicles, however, residues 6−9 of the C-terminus of BK adopt β-turn-like structures, while residues 1−5 remain “disordered”. 11,12 A similar observation was made by Glaubitz and co-workers for BK bound to the human G-protein coupled receptor B2, which was investigated by solid-state NMR: residues 6−8 (Ser-Pro-Phe) appear ordered, while residues 1−5 exhibit a high structural flexibility. 13 One potential source of conformational heterogeneity of BK, as well as of many other peptides, is proline cis−trans isomerization, 14 as the BK sequence contains proline in positions 2, 3, and 7. However, most studies performed by NMR find that in the major conformer of BK all prolyl-peptide bonds are in the trans conformation, both in free peptide and when bound to a receptor. 11−13,15−18 In general, cis−trans isomerization of the X-Pro bond is an important biological process; peptidyl-prolyl cis−trans isomerases (PPIs), which catalyze conversion between cis and trans conformations in Received: May 3, 2016 Published: July 1, 2016 Article pubs.acs.org/JACS © 2016 American Chemical Society 9224 DOI: 10.1021/jacs.6b04550 J. Am. Chem. Soc. 2016, 138, 9224−9233