Conformations of prolyl-peptide bonds in the bradykinin 1-5 fragment in solution and in the gas phase

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.

Gas-phase probes of kinetically trapped peptides

Liudmila Voronina

Gas-phase techniques are promising for studying intrinsically disordered peptides and proteins, as they can be implemented in a conformer-selective way. One of the issues that prevent them from finding a broader use is the question of to which extent the structure of a biological molecule is preserved when transferring it from solution to the gas phase via electrospray. We focus on cis/trans isomerization of prolyl-peptide bonds, which is known to have high barrier on the potential energy surface. Upon electrospraying, solution-like structures can find themselves kinetically trapped behind these barriers, as we explicitly show for the case of bradykinin 1-5 fragment. Using nuclear magnetic resonance, we determine that it adopts mostly all-trans conformation in solution, while three distinct conformational families are observed in the ion mobility experiment in the gas phase. Cryogenic ion spectroscopy combined with first-principles simulations allows us to identify the major conformers and show that the solution structural preferences of the prolyl-peptide bonds are preserved in the kinetically trapped conformational family, while the lowest-energy gas-phase structures have one of the prolines in the cis conformation. We show how collisional cross-sections and infrared spectra are used to guide and verify the calculations, giving rise to a new type of symbiosis between theory and experiment. This is especially valuable for the kinetically trapped structures, as in this case the energy criterion cannot be decisive. As a next step, we characterize the full nonapeptide bradykinin in the +3 charge state. Using field-asymmetric ion mobility spectrometry combined with cryogenic ion spectroscopy, we demonstrate the presence of three major conformational families in the gas phase, one of which is kinetically trapped. This result is in agreement with the drift-tube ion mobility data published previously, and we propose a correspondence between the conformational families separated by field-asymmetric and drift-tube ion mobility spectrometry. We obtain conformer-specific infrared spectra of the major conformers and assign the vibrational bands using 15N isotopic labeling. Substituting carbon atoms in the phenyl ring with their 13C isotope allows us to separate two types of structures according to their fragmentation pattern upon electronic excitation. This method can serve as a complementary way to introduce conformer-selectivity when studying large molecules. Finally, we assess the result of substituting one or two prolines in the bradykinin sequence with alanine. This method has been used previously to assign the peaks in the drift-time distributions to certain conformations of the prolyl-peptide bonds. We show that the mutants do reproduce a part of the conformers of the original peptide, but also form additional structures due to the higher flexibility of the alanine backbone compared to proline. We conclude that the proline-to-non-proline substitutions are helpful to assign the structures, but have to be used in conjunction with spectroscopic techniques, which allow detailed comparison of the structures of the mutant and the native peptide. In general, this work is one of the first steps towards a database of atomic-resolution peptide structures in the gas phase and a better understanding of the capabilities and limits of spectroscopy and ion mobility in the gas phase applied to intrinsically disordered peptides.

EPFL2016

Can Mutational Analysis Be Used To Assist Structure Determination of Peptides?

Chiara Masellis, Thomas Rizzo, Valeriu Scutelnic, Liudmila Voronina

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.

2018

Spectroscopic studies of kinetically trapped conformations in the gas phase: the case of triply protonated bradykinin

Thomas Rizzo, Liudmila Voronina

Understanding the relation between the gas-phase structure of biological molecules and their solution-phase structure is important when attempting to use gas-phase techniques to address biologically relevant questions. Directly after electrospray ionization, molecules can be kinetically trapped in a state that retains some “memory” of its conformation in solution and is separated from the lowest-energy gas-phase structure by barriers on the potential energy surface. In order to identify and characterize kinetically trapped structures, we have explored the conformational space of triply protonated bradykinin in the gas phase by combining field-asymmetric ion mobility spectrometry (FAIMS) with cold ion spectroscopy. We isolate three distinct conformational families and characterize them by recording their UV-photofragment spectra and vibrational spectra. Annealing of the initial conformational distribution produced by electrospray reveals that one of the conformational families is kinetically trapped, while two others are stable, gas-phase structures. We compare our results to previously published results obtained using drift-tube ion mobility spectrometry (IMS) and propose a correspondence between the conformational families separated by FAIMS and those by IMS.

Royal Soc Chemistry2015

Gas-phase probes of kinetically trapped peptides

Liudmila Voronina

EPFL2016