The Biophysical Characterization of Extracellular and Intracellular Metalloprotein Complexes

Abstract

Metals like copper and zinc play an essential role in cellular function such as enzyme cofactors, methods of electron transport, metabolic oxidation, and protein structure. However, essential metals are delicately balanced in systems to maintain homeostasis, as metal excess and starvation can both have detrimental effects. These effects include loss of enzymatic activity, oxidative stress, and protein aggregation that causes cells to become stressed and die. Copper is particularly interesting because it can cycle through multiple oxidation states, Cu+ and Cu2+, within biological systems. Metal regulation and homeostasis is not only essential to the intracellular environment but exists in the extracellular space as well. Proteins like human serum albumin and mucin glycoproteins help regulate and transport metals to cells for uptake. The goal of this work was to characterize metal binding, complex formation, and transport of extracellular and intracellular proteins to better understand metal homeostasis. To do this, human serum albumin and a segment of an intestinal mucin glycoprotein, MUC2 D1, were investigated for their ability to bind and transport Cu to a model peptide of the cellular copper importer Ctr1. Additionally, MUC2 D1 was investigated for its ability to simultaneously bind Cu+ and Cu2+ and how it reacts with a common intestinal flavonoid quercetin to begin to understand the intestinal protein’s role in copper homeostasis through complex formation. The work with extracellular proteins expands upon what is currently known regarding Cu+ transport in the extracellular environment which has been understudied and misunderstood. For the intracellular environment, glyceraldehyde-3-phosphate dehydrogenase was studied to investigate the impact of cellular copper stress on metabolic function, and periplasmic zinc-β-lactamase NDM-1 was used as a model protein to understand the propensity of ternary complexes to form with commonly used zinc fluorescent indicators. This conglomeration of extracellular and intracellular biophysical characterization shows for the first time the ability of human serum albumin to bind and transport Cu+ to a model peptide of Ctr1, MUC2 D1 to simultaneously bind multiple oxidation states of copper, and the oxidative stress that can occur to glyceraldehyde-3-phosphate dehydrogenase under copper stress and the natural defenses the cell can take to recover the damage.