Hg2+ interference with the structure of tobacco etch virus protease (TEVp) and its implications for biological engineering

Tobacco etch virus protease (TEVp) has specific and highly-conserved enzymatic activity, and is frequently applied in the fields of biology and biotechnology. Hg2+ can be utilized to purify target proteins in metal-chelate affinity chromatography. However, because Hg2+ has special biological toxity, it may endanger the stability of TEVp and hinder the utilization of TEVp in the related research. We determined the effects of Hg2+ on the structure of TEVp by means of intrinsic fluorescence, synchronous fluorescence, Fourier transform infrared (FTIR), and circular dichroism (CD) spectroscopies. The fluorescence emission and the synchronous fluorescence spectra suggested that Hg2+ induced the increased hydrophobicity around both of the tryptophan and the tyrosine residues in TEVp. Hg2+ mainly quenched the fluorescence of TEVp in a static process, with the possible formation of a complex (approximate molar ratio of 1:1), and the electrostatic force was the main binding force between TEVp and Hg2+. The FTIR and CD spectra suggested that Hg2+ induced the increased content of β-sheet in TEVp, indicating that Hg2+ may bind to the catalytic residues His46 and Cys151 in TEVp and promote TEVp to form a more compact structure. The Hg2+-induced structural changes of TEVp may potentially inhibit the catalytic activity of TEVp. These findings are beneficial for the intensive understanding of the factors that influence the stability of TEVp, and are helpful for the efficient and effective application of TEVp in the fields of biology and biological engineering.