A photochemical approach to the lipid accessibility of engineered cysteinyl residues

Abstract

Ordinary electrophilic reagents react too slowly in a nonpolar environment to be useful for the determination of the accessibility to lipid of continuous stretches of residues mutated to cysteine. By contrast, photoactivated 5-iodonaphthyl-1-azide (INA) reacted readily with 2-mercaptoethanol and dodecanethiol in nonpolar solvents and in liposomes. Continuous stretches of residues in the amphipathic N-terminal helix and first transmembrane helix of the bacterial potassium channel KcsA were replaced with cysteine, and the mutants were expressed in Escherichia coli and isolated in inner membranes. These membranes were dissolved in detergent and reconstituted into asolectin liposomes incorporating INA. The extent of light-induced reaction of INA with each cysteine was assayed by subsequent reaction with the gel-shifting, SH-specific methoxy-polyethylene glycol-2-pyridine disulfide. The pattern of apparent second-order rate constants for the photoreactions of eight substituted cysteines in the N-terminal helix conformed to other measures of lipid exposure. The pattern of the rate constants for the photoreactions of 15 cysteines in the first transmembrane helix had peaks every third residue, which partly conformed to other measures of lipid exposure.

Inactivation of Zika Virus by Photoactive Iodonaphthyl Azide Preserves Immunogenic Potential of the Virus

“…Reduction in the binding of INA-inactivated viruses with their respective antibodies has been described previously [17][18][19][20]22]. INA binds to the cysteine amino acid residues in proteins [24]. Although the lateral-ridge region of domain III (DIII) of ZIKV E-protein that binds with the ZV-54 antibody does not have a cysteine residue, there are two cysteine residues in this sequence that are present adjacent to the amino acid sequence that interacts with the antibody ZV-54 [25].…”