Electrostatic interactions of biological processes: Cellular internalization of pancreatic-type ribonucleases and salt-bridge formation in a DNA-wrapping protein

Pancreatic-type ribonucleases were studied extensively in the 20 th century and have produced a wealth of knowledge about protein structure, protein function, and enzyme catalysis. The 21st century brings a new era in which ribonucleases are being developed as potential clinical therapeutics in a wide variety of fields.;In order for a ribonuclease to be cytotoxic to cells, the ribonuclease must be internalized through endocytosis. Secondly, the ribonuclease must escape from the endosomal pathway by crossing the membrane. Once in the cytosol, the ribonuclease must be able to evade ribonuclease inhibitor (RI) in order to catalyze the degradation of RNA, which triggers the apoptotic cascade within the cell. This thesis focuses on the role of electrostatics in the internalization and endosomal escape of ribonucleases.;The three homologous members of the pancreatic-type ribonuclease superfamily studied in this thesis are Onconase (amphibian), RNase A (bovine), and RNase 1 (human). Increasing the surface positive charge of ribonucleases has been associated with increased cellular uptake and cytotoxicity. Additionally, arginine residues have been shown to be more effective than lysine residues at increasing cellular uptake of some peptides. In CHAPTER 2, the role of lysine and arginine residues in the cellular uptake and stability of Onconase is compared and contrasted through the use of a variety of experimental methods. In CHAPTER 3, the contribution of electrostatics to the binding of ONC, RNase A, and RNase 1 to model membranes is studied. By comparing experimental binding data with computational electrostatic calculations, we were able to show that whereas electrostatic forces are sufficient to explain the cellular uptake of ONC, the cellular uptake of RNase A and RNase 1 is much higher than expected based on electrostatic forces alone.;In CHAPTER 4, explicit MD simulations are used to study the effect of salt concentration on the behavior and formation of salt bridges in IHF, a DNA wrapping protein. CHAPTER 5 describes possible future directions of research regarding the future of ribonucleases as clinical therapeutics. Specifically, methods to increase cell specificity and pharmacological half-life are proposed therein.