Structural Elucidation of Biomolecular Ions in the Gas Phase Using Novel Mass Spectrometric and Computational Methods

Thesis (Ph.D.)--University of Washington, 2019Biological molecules such as protein and DNA play critical roles in various cellular reactions and carry out essential biological functions. But the mechanisms of these reactions, and the essential molecular structures to facilitate them are often poorly understood. Many efforts have been applied to these problems, but due to their complexity and various limitations of existing methods, the elucidation of biological reaction mechanisms and biological molecule or complex structures is still considered a challenge today. Therefore, the scientific community has been highly motivated to invent new methods to tackle these problems devise new angles of approach to grain insights into biomolecular structure. This dissertation summarizes some recent work in characterizing cation-radical reactions, and structures of gas-phase molecular complexes using novel mass spectrometry methods in combination with theoretical computational modeling. The novel mass spectrometric methods presented in this work are gas-phase photo-dissociative crosslinking techniques and UV-visible photodissociation action spectroscopy. They have several unique advantages in tackling structure elucidation of weakly bound complexes and transient radical species. 1) The mass spectrometer (MS) is a universal detector which is widely used to characterize various kinds of biological molecules. 2) MS works with ions of interest that are generated and stored in the gas phase at low pressure of an inert gas (He). It is the perfect system to study reaction mechanisms because of low interference from the ambient environment, such as solvent, counterions, surfaces, etc. 3) Methods exist to generate cation radicals in the gas phase, and thanks to the low pressure inside a mass spectrometer, the produced species are kinetically stable on the experimental time scale of several milliseconds. 4) MS is a perfect tool for conducting gas-phase reactions because it allows one to manipulate the ion population and select and focus the ions. It is advantageous for crosslinking reactions that often suffer from low concentrations of reactants when the reaction is attempted in condensed phase. 5) The experimental action spectra can be interpreted using sophisticated computational techniques providing vibronic transitions of multiple isomeric candidates. The new data generated with these new mass spectrometry methods offer unique insights, but also pose challenges to experimental data interpretation. Various computational approaches are used to supplement the experimental data, and the results from the computations are used to guide the interpretation. This dissertation also includes several novel approaches on the computational front, introducing the customized modeling pipeline with a combination of Born-Oppenheimer dynamics, density functional theory calculations, and machine learning techniques. The first chapter introduces some basic terms and outlines the background for the topic of study, including a quick review of the current challenges and techniques in the field of structure elucidation of biomolecules. Also introduced are the fundamentals of mass spectrometer, commonly used and newly developed ion activation methods, and computational modeling. This chapter lays the foundation of the work described in later chapters. A small neuroprotective peptide Cys-Ala-Gln-Lys (CAQK) has recently been discovered to mitigate adverse effects of brain trauma in mice, possibly because of specific interactions with yet-to-be identified proteins. In Chapter 2, an application of photo-crosslinking is described to study the noncovalent interactions of CAQK with several model target peptide motifs. The experimental results in combination with Born–Oppenheimer molecular dynamics revealed the structural preferences for binding to the amino acid residues of potential target peptides. In Chapter 3, inspired by the biological significance of CAQK, we employed the photo-dissociative crosslinking techniques to probe its interactions with several dinucleotides as structure motifs of nuclear DNA. This lysine-containing peptide can be viewed as a simplified surrogate of a histone interacting with DNA. We were able to show that in interactions with CAQK ions, even simple dinucleotides differ in their binding efficiency and stereochemistry. We provided structures of selected complexes obtained by electronic structure theory calculations using density functional theory (DFT). UV and energetic particle radiation can ionize DNA creating electron deficiency (hole) at nucleobases. These holes can migrate along the strand, leading to lesions and DNA damage by follow up radical reactions. Chapter 4 and chapter 5 are focused on the characterization of DNA cation radicals in the guanine and cytosine containing dinucleotide as model systems in probing the electron transfer mechanisms in DNA radiation damage. Experimental action spectra were obtained for these small nucleotides and the absorption bands were interpreted by finding the closest match from the calculated spectra. The last chapter features an ongoing project in which we have made an attempt, using photo-crosslinking techniques, to probe noncovalent interactions within a complex consisting of a chiral agonist and its binding motifs. The experimental results revealed high binding affinity of the native agonist regardless of chirality, however the crosslinking fragment was not observed. Several DFT optimized low energy structure had shown a consistently low “contact” rate of the phototag with the target peptide. A further characterization of ion structures will be complemented from the collisional cross section analysis by ion mobility (IM) measurements