Can Mutational Analysis Be Used To Assist Structure Determination of Peptides? Liudmila Voronina, Valeriu Scutelnic, Chiara Masellis, and Thomas R. Rizzo* Laboratoire de Chimie Physique Mole ́ culaire, E ́ cole Polytechnique Fe ́ de ́ rale de Lausanne, EPFL SB ISIC LCPM, Station 6, CH-1015 Lausanne, Switzerland *S Supporting Information ABSTRACT: Mutational analysis is widely used to study the relationship between sequence and structure of proteins and peptides. It is often assumed that substituting a proline with another amino acid “locks” the peptide bond in the trans conformation, allowing only a subset of the initial molecular geometries to be observed. To test this assumption, we assess the result of substituting two prolines in the bradykinin sequence with alanine using field-asymmetric ion mobility spectrometry combined with cryogenic ion spectroscopy in the gas phase. While the structure of the mutant coincides with a part of the conformational space of the original peptide, the higher flexibility of the alanine backbone compared to proline allows it to access additional structures. We conclude that proline-to-nonproline substitutions are helpful to assign structures, but they should be used in conjunction with spectroscopic techniques that allow detailed comparison of the structures of the mutant and the native peptide. S ubstituting an amino acid in a protein sequence with another residue is often referred to as mutational analysis. 1 This procedure reveals how critical a certain amino acid is for the structure and function of a protein. In particular, it has been used to study the role of proline as the only canonical amino acid for which cis and trans isomers of the peptide bond are comparable in energy. 2 To assess the role of rare cis states (5.2% according to Weiss and co-workers), 3 the possibility of isomerization can be eliminated by “locking” the Xxx-Pro bond to the trans isomer via substituting the proline with another amino acid. 4,5 Mutational analysis, in conjunction with various methods of structural determination, shows that proline performs multiple functions within the protein sequence: It acts as a molecular switch, 6,7 and its isomerization is often a rate-limiting step in protein folding. 8,9 In order to deepen the understanding of the structural role of proline, one can isolate a model system from its native environment and apply powerful gas-phase tools that can be implemented in a conformer-selective way. For example, it was suggested that proline is one of the primary reasons for the formation of distinct conformational families of short peptides in the gas phase, resulting in multiple peaks in their collisional-cross section (CCS) distributions determined by ion mobility spectrometry (IMS). 10−13 In order to establish a correspondence between the features in the CCS distribution and conformational preferences of prolyl-peptide bonds, the same point mutations as in solution-phase studies have been used. 11,14−16 In the case of triply protonated bradykinin (BK), for instance, three conformational families in the gas phase were attributed to different cis−trans isomers using proline-to- alanine substitutions. 15,17 It has been determined that protein secondary structure is influenced by the presence of proline in two somewhat orthogonal ways. On the one hand, proline disrupts helix and β- sheet formation and increases the level of backbone disorder. 18 On the other hand, it is conformationally restricted and makes the protein backbone more rigid. 19 If proline is substituted by another amino acid, the resulting structure depends on the interplay between these factors. Neverthless, mutational analysis is often based on the assumption that upon “locking” a peptide bond in the trans conformation, only a subset of the initial molecular geometries should be observed. 1,4,7,15,17,20−22 In this work we test this assumption for a mutant of bradykinin using a combination of ion mobility spectrometry and double- resonance, cryogenic-ion spectroscopy in the gas phase. The experimental procedure is similar to that used previously 23 and is described in the Supporting Information. Briefly, we generate gas-phase ions by electrospray, separate them into conformational families using field asymmetric ion mobility spectrometry (FAIMS), 24 and inject them into a home-built spectrometer, where we perform spectroscopic studies at cryogenic temperatures. 25 We have demonstrated previously that FAIMS allows us to partially separate several conformational families of BK 3+ (Figure 1a). 23 Using cryogenic-ion spectroscopy as a detector for a specific conformation, we observe that BK 3+ forms at least three conformational families in the gas phase, which we denoted I, II, and III. 23 In this study we focus on kinetically trapped conformational families I and II observed directly after ESI with low collisional activation and preserved upon FAIMS separation. Normal bradykinin includes prolines in positions 2, 3 and 7, and the barriers for cis−trans isomerization are considered to be the origin of distinct conformational families in the gas phase. 15,17 Here we explore the conformational space of triply protonated Pro3&7 → Ala mutant of BK with proline in position 2. Pierson and co-workers suggested that this mutant reproduces the structure of conformational family B of native BK, separated by the drift tube IMS. 17 Received: October 23, 2017 Published: February 7, 2018 Communication pubs.acs.org/JACS Cite This: J. Am. Chem. Soc. 2018, 140, 2401-2404 © 2018 American Chemical Society 2401 DOI: 10.1021/jacs.7b11302 J. Am. Chem. Soc. 2018, 140, 2401−2404 Downloaded via ECOLE POLYTECHNIC FED LAUSANNE on May 6, 2019 at 12:44:16 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.