Carboxyl group-modified myoglobin shows membrane-permeabilizing activity

In this study, we investigated whether modification of the carboxyl group with semicarbazide-enabled myoglobin (Mb) exhibits membrane-perturbing activity in physiological solutions. Mass spectrometry analysis showed that semicarbazide molecules were coupled to 19 of the 22 carboxyl groups in semicarbazide-modified Mb (SEM-Mb). Measurements of the absorption and circular dichroism spectra indicated that SEM-Mb lost its heme group and reduced the content of the alpha-helix structure in Mb. The microenvironment surrounding Trp residues in Mb changes after blocking negatively charged residues, as shown by fluorescence quenching studies. The results of the trifluoroethanol-induced structural transition indicated that SEM-Mb had higher structural flexibility than that of Mb. SEM-Mb, but not Mb, induced the permeability of bilayer membranes. Both proteins showed similar lipid-binding affinities. The conformation of SEM-Mb and Mb changed upon binding to lipid vesicles or a membrane-mimicking environment composed of SDS micelles, suggesting that membrane interac-tion modes differ. Unlike lipid-bound Mb, Trp residues in lipid-bound SEM-Mb are located at the protein-lipid interface. Altogether, our data indicate that modifying negatively charged groups relieves the structural con-straints in Mb, consequently switching Mb structure to an active conformation that exhibits membrane-permeabilizing activity.

Automated summary

This study investigates the membrane-perturbing activity of semicarbazide-modified myoglobin (Mb) in physiological solutions. The results show that semicarbazide modification leads to structural changes in Mb, including loss of heme group and reduced alpha-helix content. SEM-Mb exhibits higher structural flexibility and membrane permeability compared to Mb, suggesting that modification of negatively charged groups relieves structural constraints in Mb, leading to its active conformation with membrane-permeabilizing activity.