Development of scattering correction and UV spectral simulation routines to facilitate investigations on the inhibitory metal site in anthrax lethal factor via tyrosine modification

Abstract

Anthrax lethal factor (LF) is a zinc-dependent metalloendopeptidase and critical protein component of the anthrax toxin. Previous studies have shown that LF possesses an inhibitory metal binding site which is involved in accelerating metal exchange reactions via an associative mechanism. The current study sought to employ chemical modification strategies in combination with UV-Vis spectroscopy to assess whether a tyrosine residue (Tyr728) near the active site of LF is involved in binding the inhibitory metal ion and in participating in metal exchange. Tyr residues in LF were successfully modified with N-acetylimidazole, however, complications arose in the course of their quantification. For instance, the emergence of variable light scattering complicated the spectrophotometric analysis of the protein samples. To address this, an Excel-based tool was developed that allows for the correction of protein spectra, taking into account both Rayleigh and Mie scattering contributions. Furthermore, the degree of Tyr modification varied drastically (from 5 to 13 modified Tyr residues) for samples prepared under identical experimental conditions when protein concentrations were calculated via a single-wavelength approach. Hence, a second Excel- based routine was designed with the view of calculating more accurate protein concentrations by determining the molar absorption coefficients at each wavelength between 250 and 350 nm. This was achieved by simulating protein spectra based on the abundance and pseudo-Voigt profiles of their amino acid constituents. By implementing this multi-wavelength fitting approach, a better estimate of the number of modified Tyr residues was obtained. Together, the tools produced in this work facilitate the accurate determination of protein concentrations not just for LF, but for proteins in general.