Publication
Proteins and even small peptides play crucial role in almost any biological. Biological activity and functionality of these building blocks of life are determined not only by their primary structure, but also by their 3D orientation â secondary, tertiary and quaternary structural motifs. This structure-function relationship is one of the most important paradigms in structural biology and makes characterization of the geometry of biological molecules an extremely important task. In contrast to experimental techniques in condensed phase Cold Ion Spectroscopy studies biomolecular ions isolated in the gas phase and provides vibrational fingerprints specific for each conformer, allowing a direct comparison with high-level calculations. The method, widely known as â double-resonanceâ spectroscopy, has several fundamental and practical limitations and is applicable only for peptides smaller then 10 - 15 amino acids, necessarily containing at least one of aromatic amino acids and having vibrationally resolved electronic spectrum. The main goal of this thesis is development and demonstration of a novel conformer-selective spectroscopic technique, IR-IR-(D)UV hole-burning spectroscopy. This multi-laser experiment has an important advantage over the double-resonance approach, as it does not depend on the shape or resolution of the electronic spectrum of peptide, and allows extending the application of Cold Ion Spectroscopy to peptides with any amino acid composition. The first part of this work describes application of this technique for recording the conformer selective vibrational spectra of protonated aromatic amino acids tryptophan and histidine, for which the double-resonance cold ion spectroscopy was precluded due to their unstructured electronic spectra. Combined with high-level anharmonic calculations, conformer specific vibrational spectra allowed solving structures of gas-phase conformers of these biomolecular ions. The second part describes the use of peptide bond as an alternative natural chromophore, present in any peptide. The electronic spectra of peptide bonds in cold protonated peptides in the gas-phase as well as the influence of an IR pre-excitation are explored in details. The use of peptide bonds allows recording high-quality linear vibrational spectra of peptides even without aromatic amino acids, for which only IRMPD and tagging spectroscopies were available until recently. This feature makes IR-IR-DUV hole-burning spectroscopy the most universal conformer-selective spectroscopic tool. The third part of this thesis is dedicated to the relationship of gas-phase and native structures of biomolecular ions. The lack of solvent in the gas-phase mimics to some extent hydrophobic media of cell membranes or receptors, where intramolecular hydrogen bonds rather than interaction with the solvent largely determine the structure of peptides. In such cases general structural motifs can survive the transfer to the gas phase. Experimental data