Cholesteryl-Conjugated Ribonuclease A Exhibits Enzyme Activity in Aqueous Solution and Resistance to Dimethyl Sulfoxide

Abstract

Using bovine pancreatic ribonuclease A (RNase A) and cholesterol, we synthesized cholesteryl-conjugated ribonuclease A (CHRNase A) to evaluate the influence of a conjugated hydrophobic moiety on protein function. Nuclear magnetic resonance and matrix-assisted laser desorption/ionization time-of-flight spectrometry suggested that one cholesteryl group was conjugated to RNase A. Differential scanning calorimetry indicated that CHRNase A was denatured in the solid state but was folded in phosphate buffer (0.05 mol/L, pH 6.5). CHRNase A resembled RNase A in its secondary structure, but circular dichroism (CD) spectra revealed that the helical content of CHRNase A was decreased and the tertiary structure of CHRNase A differed from that of RNase A. Furthermore, fluorescence measurements, CD spectra, an 8-anilino-1-naphthalenesulfonic acid ammonium salt-based assay, and surface tension measurements suggested that cholesterol was conjugated to a tyrosine residue on the protein surface. The relative activity of CHRNase A to RNase A was 79 ± 7%, and the enzyme activity of CHRNase A by adding β-cyclodextrin (β-CyD) increased to 129 ± 7%. Therefore, we considered that the cholesteryl group interacted with substrate (cytidine 2′3′-cyclic monophosphate monosodium salt) to inhibit the enzyme reaction. Finally, the environment around tyrosine residues in CHRNase A in dimethyl sulfoxide was similar to that of native RNase A in phosphate buffer (0.05 mol/L, pH 6.5). These results suggest that cholesterol conjugation to RNase A altered RNase A functionality, including improvement of RNase A resistance to dimethyl sulfoxide and modulation of the ability of β-CyD to control RNase A enzymatic activity.

Unfolding of the SARS-CoV-2 spike protein through infrared and ultraviolet-C radiation based disinfection

“…Based on the obtained optimized conditions, the effect of treatment on the secondary structures of SARS-CoV-2 spike protein and another model protein RNase A was studied. RNase A is a structurally versatile and highly stable protein having 124 amino acid residues [24] , [25] . The main focus of the present work is to provide an inexpensive and efficient disinfection method by combining IR and UV-C irradiations to fight against contagious infections by viruses and bacterial pathogens.…”