Exploring and Exploiting Ribonuclease 1: from Protein Biochemistry to Protein Engineering

Abstract

Ribonuclease (RNase) 1 is a human protein with a remarkable ability to indiscriminately hydrolyze RNA. RNase 1 and its bovine homologue RNase A exhibit ubiquitous expression across tissues, a catalytic efficiency within the diffusion-limited regime, and minimal substrate sequence requirements. RNase A has been a favorite model protein of biochemists for over half a century; due to the high level of sequence conservation between RNase A and RNase 1, many observations made for RNase A have corollaries for RNase 1.

RNase 1 and RNase A are members of the pancreatic-type ribonuclease (ptRNase) superfamily, a class of enzymes which share many biophysical features, including a small molecular weight, high cationicity, and a secretory nature. Historical elucidation of ribonuclease biochemistry describes their susceptibility to oxidation-induced inactivation. This raises the question: how are these secretory enzymes able to preserve catalytic competency in oxidatively challenging extracellular environments such as blood serum and even epidermal skin?

In Chapter 2 of this thesis, the intrinsic antioxidative capacity of RNase 1 is described. Chemical biology and biomimetic techniques corroboratively implicate two methionine residues as sacrificial antioxidants to protect the enzymic active site, allowing catalysis to persist in the presence of reactive oxygen species. In silico studies suggest evolutionary patterns to install these antioxidative features across the ptRNase superfamily. Sulfur–arene interactions appear to tune the reactivity of methionine residues in a manner consistent with rates of oxidation. These findings highlight an underappreciated role for methionine—to protect catalytic histidine residues—and indicate a means by which ptRNases remain functional in oxidatively challenging physiological environments.

The desirable biophysical features of RNase 1 and the wealth of biochemical knowledge regarding it have also made it a favored model system of protein engineers, as exemplified by RNase S and cyclic RNase-based zymogens, two systems which reversibly attenuate ribonucleolytic activity. In particular, RNase-based zymogens can be activated by exogenous proteases; this schema has biotherapeutic potential, as demonstrated by zymogens which activate in response to viral infection and exert cytotoxic ribonucleolytic activity.

Efforts to establish a zymogen directed toward the coronavirus SARS-CoV-2 are described in two parts of this thesis. In Chapter 3, the main protease 3CLpro of SARS-CoV-2 is enzymologically characterized. This work clarifies reported inconsistencies in enzymological features of this key viral protease and relies on a non-Michaelis–Menten, Bayesian inference-based analytical technique to circumvent some of the causes of the inconsistent prior reports. Then, in Chapter 4, the newfound knowledge of 3CLpro enzymology is applied toward the design of an RNase 1-based, 3CL superscript pro - directed zymogen. The zymogen is inactivated by steric occlusion and conformational distortion of the active site, and site-specific activation by 3CL superscript pro results in a multi-order of magnitude increase in ribonucleolytic activity. 3CL superscript pro action upon the zymogen leads to ribonucleolytic turnover of a fluorescent RNA substrate by the activated species, affording signal amplification that enables detection of nanomolar 3CLpro concentrations in a timeframe comparable to rapid antigen detection testing.