Structure, folding, and dissociation of gaseous biomolecules

A multitude of noncovalent interactions is responsible for the three-dimensional structure and the free energy surface for folding of a biologically active molecule, including its interactions with solvent. In solution phase experiments, however, it is very difficult, if not impossible, to separate the contribution of solvent from intrinsic biomolecular stability. A new approach in addressing the effect of external solvation is to completely remove any solvent, and study the structure and energetics of the biomolecule by itself. To what extent and on what time scale native biomolecular structures can be retained in the gas phase, and which intramolecular interactions could be responsible for (transient) structural stabilization in a solvent-free environment, will be studied here in native electron capture dissociation (NECD) experiments. However, in the gas phase, the biomolecular ions are expected to undergo structural rearrangements that eventually lead to stable gas phase structures. These will be studied here in detail in electron capture dissociation (ECD) and electron detachment dissociation (EDD) experiments, from which structural data as well as thermodynamic parameters (enthalpy, entropy, free energy) for the folding/unfolding equilibrium will be obtained to gain deeper insight into the structure and the dominant forces in gaseous ion folding. Furthermore, the recently developed EDD technique will be explored here as a new tool for the probing of nucleic acid structure. In another part of this project, a new approach for post-charging of biomolecular ions from electrospray ionization (ESI) will be investigated. The added charge could substantially improve mass spectrometric analysis of the biomolecular ions for a number of reasons. First, adding charge increases Coulombic repulsion, which aids in the unfolding of the gaseous ion`s tertiary structure that could otherwise prevent dissociation in tandem mass spectrometry experiments. Second, this could produce narrower molecular ion charge distributions, which in turn would reduce the charge heterogeneity of the fragment ions and facilitate their detection (fewer signals of higher signal-to-noise ratio). Third, adding charge increases the signal-to-noise ratio in mass spectrometers with charge- sensitive detection. Fourth, producing more highly charged ions is advantageous for sequencing with ECD as the probability for electron capture increases with the square of the ion`s charge. Fifth, gaseous biomolecular unfolding by post-charging instead of ion heating avoids loss of labile post-translational or -transcriptional modifications.

In this project, modern dissociation techniques were used to address fundamental research questions regarding biomolecular structure and folding, and to develop new methodology for biomolecular sequencing. In one part of the project, the effect of desolvation on the structure of gaseous proteins was studied, and it was found that the overall stability of a protein fold in solution is not correlated to its stability in the gas phase. For example, the protein Cytochrome c is highly stable in solution but unfolds on a milliseconds timescale after transfer into the gas phase, whereas the native fold of the moderately stable protein KIX is preserved in the gas phase on a seconds timescale, and undergoes only partial unfolding upon heating that causes breaking of covalent bonds. In another part of the project, we studied the process by which proteins fold into biologically active structures in the first place, and found that solvent water not only makes possible fast but also correct folding. These new insights are important for understanding how different chemical environments, e.g., the cytosol or membranes, affect protein structure and stability. Ribonucleic acids (RNA) are another class of biomolecules that are indispensable to life, and many of their functions beyond translating the DNA (deoxyribonucleic acids) code into proteins are only beginning to be explored. These so-called non-protein coding RNAs frequently carry posttranscriptional modifications that are essential to the specific tasks that they fullfill, but at the same time, these modifications can render RNA sequencing by next generation methods virtually impossible. Here we have developed a new mass spectrometry based approach for de novo sequencing of highly modified RNA that relies on the use of two different dissociation techniques, one of which involves a complex mechanism based on radical ion chemistry. Moreover, we have studied the mechanism involved in RNA dissociation by mere ion heating and found that it proceeds by a stepwise mechanism similar to the transesterification and cleavage steps involved in RNA hydrolysis in solution. Our straightforward approach can identify and localize both posttranscriptional and synthetic modifications, and is applicable to RNA consisting of ~80 nucleotides, including transfer RNA.