Structural and analytical identifications of biomolecules by cold ion spectroscopy of non-covalent complexes

Non-covalent interactions are ubiquitous in nature. The diversity and functionality of these interactions are employed by nature as a universal tool for building the molecular mechanism of life. Although weak, these interactions play a key role in both formation of 3D structures of biomolecules through intra- and intermolecular binding and highly selective coupling between biomolecules. In this thesis, we explore these interactions in two directions: (i) for solving native 3D structures of biomolecules and (ii) for analytical identifications of isomeric carbohydrates. Both studies use the technique of cold ion spectroscopy (CIS), which often enables vibrational resolution in infrared and electronic spectra of biomolecules isolated in the gas. Although the resolved spectra can be used in solving the intrinsic molecular 3D structures of biomolecules, life science research desperately needs their native structures. It is challenging however to get vibrational resolution in solution phase, where these structures originally reside. As a compromise, we do not fully dehydrate the biomolecules during their gentle electrospray ionization (ESI) and leave a few water molecules attached to the ion. The structure of such microhydrated molecule may resemble its native one in solution while working in the gas phase enables the desired vibrational spectral resolution.The second direction of the study aims to explore the sensitivity of intermolecular noncovalent interactions in biomolecular complexes to fine structural details of the partners. The work demonstrates how this sensitivity can be used for analytical identifications of the biomolecules that are highly rich in isomers.In the first part of this work, we report on the elucidation of native structures of small protonated biomolecules glycine and triglycine. We apply cold ion IR spectroscopy to the gas-phase complexes of these biomolecules, in which they are microhydrated by a controlled number of water molecules. The complexes were produced directly from an aqueous solution, using a gentle ESI source. Our studies suggest that retaining molecules in the complexes generated from the solution allows for preserving the main structural features of the native structures.In the second part of the thesis, we report on the investigation of the sensitivity of non-covalent interaction between aromatic molecules and carbohydrates to structural details of binding partners. We calculated structures of non-covalent complexes of aromatic molecules with isomeric monosaccharides and validated them by IR spectroscopy. It appears, that the change of an analyzing isomeric carbohydrate molecule significantly influences the UV absorption of the aromatic sensor molecule. The spectral difference between the complexes is explained by the interplay of different intermolecular non-covalent bonds, which may not be all identical for different isomers. Even structural changes in the groups that are not directly involved in non-covalent binding with chromophore exhibit a significant impact on UV spectra.In the last part of the thesis, we explore the use of non-covalent carbohydrate-aromatic complexes for the identification of isomeric glycans using a unique method of 2D UV-MS CIS. We demonstrated the applicability of this method for accurate identification and quantification of all types of isomeric carbohydrates and tested the performance of the technically simpler version of the method, 1D UV fragmentation spectroscopy.

Cold ion spectroscopy for structural identifications of biomolecules

Oleg Boyarkine

Over the last decade, the spectroscopy of cryogenically cold ions iso- lated in the gas phase has been developed as a new tool for structural elucidations of biological molecules. Cooling allows for vibrational resolution in UV and IR spectra of small to midsize peptides, enabling different multi-laser techniques of conformer-specific spectroscopy. In conjunction with quantum chemistry calculations, IR spectra of sin- gle conformers allows for solving their intrinsic geometries. Here, we briefly review some fundamental and technical aspects of the cold- ion spectroscopy (CIS) approach and illustrate its application for pro- tonated peptides, carbohydrates and for non-covalent complexes of biomolecules. The challenges and limitations of CIS in view of its rele- vance to life-science studies are critically assessed. Finally, we discuss and illustrate some approaches of CIS for the analytical identification of biomolecules, in particular the recently developed method of 2D UV-MS fingerprinting, which combines CIS with high-resolution mass spectrometry.

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

Cold Ion Spectroscopy of Biological Molecules in the Gas Phase

Natalia Nagornova Boyarkine

The three-dimensional (3D) structures of proteins and peptides in vivo largely determine their biological functions. In vitro these native structures and their heterogeneity reflect a subtle balance between noncovalent intramolecular interactions and those with the surrounding solvent molecules. Decoupling the intra- and intermolecular interactions and revealing the intrinsic structures of biomolecules in the gas phase is crucial for understanding protein-peptide (-protein, -membrane) binding processes and protein folding, and can assist in silico drug design. In addition to this, the spectroscopic studies of (bio)molecules in the gas phase provide crucial information that serves as a benchmark for validation of theoretical approaches, used for structural computations. In this thesis report we demonstrate the use of conformer-selective, Cold Ion Spectroscopy (CIS), combined with mass spectrometry and electrospray ionization technique, to reveal spectroscopic fingerprints of closed-shell (bio)molecular species in the gas phase. These fingerprints provide a stringent test for validating calculated structures of the molecules. Electronic and conformer-specific vibrational spectra of charged molecules, collisionally cooled in a linear radio frequency 22-pole ion trap have been measured using photofragment detection schemes that employs UV and/or IR laser light. A detailed set of spectroscopic and structural constraints, obtained using CIS method for an antibiotic peptide, gramicidin S (GS) allows an unambiguous determination of its 3D structure in the gas phase. The calculated gas-phase structure of GS differs substantially from its known structure in the condensed phase and, thus, provides complementary information for modeling biological activity of this antibiotic. At the next step we form hydrogen-bonded complexes of GS with a gradually increasing number of water molecules. Spectroscopy of such peptide-water clusters targets to bridge the gap between the gas phase and the in vitro structures. We have shown that both, UV and IR spectra of large GS-water clusters remain vibrationally resolved, if the species are cooled to cryogenic temperatures. The observed detailed spectroscopic fingerprints of the hydrated peptides provide a benchmark for their structural analysis. While CIS method was developed for biological species, here we demonstrate its successful application for structural determination of highly-reactive catalytic metalorganic species. The experimentally observed lack of a characteristic NH stretch vibration in the IR spectra of such a species allowed an unambiguous selection of its structures from several low-energy calculated candidates. The current approach for solving structures of biomolecules implies a comparison of a measured spectroscopic fingerprint with that, calculated for a candidate structure. Unfortunately, high accuracy structural computations become time consuming and challenging for large peptides. We revealed a specificity in electronic spectra of phosphorylated peptides, which coordinates with the docking position of a phosphogroup in peptides. This observation, if confirmed for a large variety of peptides, may allow a prediction of phosphorylation sites without a call for calculations. Regarding a relative simplicity of UV spectroscopy we see a potential of this approach as an orthogonal analytical method that assists mass spectrometry in some puzzling cases of peptide analysis. At the end of this work we tried to push the limits of CIS approach towards very large species and, for the first time, measured IR spectrum of a bare protein, cytochrome c, in the gas phase.

EPFL2011

Cold Ion Spectroscopy of Biological Molecules in the Gas Phase

Natalia Nagornova Boyarkine

EPFL2011

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

Caroline Seaiby

EPFL2011