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

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.

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

Combining Ion Mobility and Cryogenic Spectroscopy for Structural and Analytical Studies of Biomolecular Ions

Michael Zachary Kamrath, Thomas Rizzo

Ion mobility spectrometry (IMS) has become a valuable tool in biophysical and bioanalytical chemistry because of its ability to separate and characterize the structure of gas-phase biomolecular ions on the basis of their collisional cross section (CCS). Its importance has grown with the realization that in many cases, biomolecular ions retain important structural characteristics when produced in the gas phase by electrospray ionization (ESI). While a CCS can help distinguish between structures of radically different types, one cannot expect a single number to differentiate similar conformations of a complex molecule. Molecular spectroscopy has also played an increasingly important role for structural characterization of biomolecular ions. Spectroscopic measurements, particularly when performed at cryogenic temperatures, can be extremely sensitive to small changes in a molecule’s conformation and provide tight constraints for calculations of biomolecular structures. However, spectra of complex molecules can be heavily congested due to the presence of multiple stable conformations, each of which can have a distinct spectrum. This congestion can inhibit spectral analysis and complicate the extraction of structural information. Even when a single conformation is present, the conformational search process needed to match a measured spectrum with a computed structure can be overwhelming for peptides of more than a few amino acids, for example. We have recently combined ion mobility spectrometry and cryogenic ion spectroscopy (CIS) to characterize the structures of gas-phase biomolecular ions. In this Account, we illustrate how the coupling of IMS and CIS is by nature synergistic. On the one hand, IMS can be used as a conformational filter to reduce spectral congestion that arises from heterogeneous samples, facilitating structural analysis. On the other hand, highly resolved, cryogenic spectra can serve as a selective detector for IMS that can increase the effective resolution and hence the maximum number of distinct species that can be detected. Taken together, spectra and CCS measurements on the same system facilitates structural analysis and strengthens the conclusions that can be drawn from each type of data. After describing different approaches to combining these two techniques in such a way as to simplify the data obtained from each one separately, we present two examples that illustrate the type of insight gained from using spectra and CCS data together for characterizing gas-phase biomolecular ions. In one example, the CCS is used as a constraint for quantum chemical structure calculations of kinetically trapped species, where a lowest-energy criterion is not applicable. In a second example, we use both the CCS and a cryogenic infrared spectrum as a means to distinguish isomeric glycans.

2018

Spectroscopic Probes of Conformational Isomerization of Biological Molecules in a Cold Ion Trap

Caroline Seaiby

The function of biologically active molecules depends both on their structure and on their conformational dynamics. In solution, intramolecular interaction with the solvent will influence different biological processes, i.e. protein folding. However secondary structure (helices, sheets) is heavily influenced by intramolecular forces and hence it is important to isolate biological molecules in order to study their intrinsic behavior. Moreover, gas phase studies on biomolecules provide critical information that can serve as benchmarks to test the accuracy of theoretical predictions. The main focus of this thesis is the investigation of the potential energy surfaces of biomolecules in the gas phase. Biomolecular ions are produced in the gas phase via nano-electrospray, mass-selected and guided into a cold, 22-pole ion trap where they are cooled via collisions with cold helium. A variety of double-resonance techniques based on photofragmentation detection are then applied to obtain information on the molecule stable conformations, the potential barriers separating stable conformations and their connectivity. We applied these techniques on molecules of increasing size, starting with protonated phenylalanine and proceeding to the 7amino acid peptides, Ac-Phe-(Ala)5-LysH+ and the 12-residue peptide Ac-Phe-(Ala)3-(Gly)4-(Ala)3-LysH +. As a first step, electronic spectra and conformation-specific IR-UV double resonance spectra are measured. These measurements, in combination with DFT calculations, allow the assignment of two stable conformers in the case of the protonated phenylalanine and four in the case of the seven residue peptide. In the second part of this work we focused on the conformational isomerization of these molecules by performing infrared and ultraviolet hole-filling spectroscopy in the ion trap. We demonstrated that we can induce isomerization between stable conformers of each molecule via vibrational excitation. After energy dissipation, the excited molecules are redistributed among the initially identified conformers, but no new minima were detected. In the last part of this thesis, the fractional populations and the isomerization quantum yields are determined through infrared induced population transfer spectroscopy. Protonated phenylalanine reveals conformational selectivity in its isomerization while the relaxation of the infrared excitation leads to the equilibrium distribution in the case of the 12-residue glycine-containing peptide. The steps that occur during the energy dissipation are discussed in this thesis.

EPFL2011