UC Davis

The works included in this dissertation focus on the application of analytical and biochemical methodologies to study metal-binding and metal-regulating biomolecules. Additionally, aspects of molecular biology, programming, and spectroscopy are found in these works. This thesis describes the implementation of mass spectrometry-based techniques to understand metal dynamics in the extracellular space. Chapter One describes the current state of mass-spectrometry-based applications toward the discovery of metal-binding species and methods to determine metal-interaction sites. This chapter highlights techniques for understanding metal dynamics of metal-binding biomolecules as well as monitoring the distribution of metal micronutrient speciation in the extracellular space. Topics include sample preparation, native and non-native mass spectrometry (NMS), size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICP-MS), and immobilized metal affinity chromatography (IMAC). This chapter will provide the framework for the following chapters in this thesis. Chapter Two describes efforts to elucidate the zinc and copper binding propensity and binding site of the peptide hormone C-peptide. Methods used include mass spectrometry (MS), nuclear magnetic resonance (NMR), ultraviolet–visible light spectroscopy, and isothermal calorimetry (ITC). These studies determined that Cu2+ binding occurs primarily at the N-terminus; however, other interaction sites beyond what could be confidently detected may occur at the C-terminal region. Chapter Three focuses on identifying copper-binding peptides by IMAC. This chapter includes work towards the development and the utilization of IMAC to enrich synthetic metal binding peptides as well as the peptide hormone hepcidin – showing possible structural dependence of Hepcidin for Cu2+ mediated ternary complex formation. IMAC work was further extended to identify Cu2+ binding peptides from enzymatically digested rice bran. Specific peptides were selected based on common sequence motifs and hydrophobicity. Selected peptides were investigated for their Cu2+ binding and anti-oxidative properties through chelator, copper reduction, and copper reactivity assays. Lastly, we demonstrate through in vitro studies followed by western blot increased AMP-activated protein kinase (AMPK) activation for each peptide, indicating a link between altered copper bioavailability and decreased diabetic complications. Chapter Four includes developing and optimizing an IMAC procedure for enriching copper-interacting proteins from serum and plasma. This work describes buffer optimization, rational chelator selection, serum loading considerations, and applications for probing biological states. Specifically, this chapter describes the use of IMAC to investigate the strength of copper-mediated ternary complex formation of serum protein, revealing differences concerning the amount of serum applied to a fixed amount of resin. Further, work involving the analysis of plasma collected from patients with Wilson disease and without using a traditional proteomics workflow and the developed IMAC-proteomics workflow is described. Preliminary data from applying IMAC coupled with proteomics to enrich metal binding species from serum collected from obese and post-bariatric surgery mice. This work chapter showcases the use of IMAC beyond the identification of metal interacting species and provides evidence of changes in metal-binding propensities in disease states. Chapter Five describes collaborative work towards understanding monosaccharides’ influence in the copper-containing ferroxidase ceruloplasmin. Previous work has demonstrated that ceruloplasmin concentration is decoupled to its bioactivity in the presence of monosaccharides such as glucose. Moreover, in the presence of ceruloplasmin degrades at a higher rate through an unknown mechanism. This chapter described our efforts to both substantiate these observations and elucidate the mechanism of glucose-dependent bioactivity.