Molecular characterization of myoglobin from Sciurus vulgaris meridionalis: Primary structure, kinetics and spectroscopic studies

Abstract

Myoglobins (Mbs) are heme-proteins involved in dioxygen storage necessary for metabolic respiration. Mbs are intensely investigated as archetype to investigate structure/function relationship in globular proteins. In this work, the myoglobin from Sciurus vulgaris meridionalis has been for the first time isolated and purified with a high yield and homogeneity. The primary structure characterization has been performed by applying a strategy based on high resolution tandem mass spectrometry. Proximal (position 93, α-helix F8) and distal (position 64, α-helix E7) histidinyl residues as well as most of the amino acid residues (i.e., Leu29, Lys45, Thr67, Val68) involved in the autoxidation mechanism are conserved in the squirrel Mb. The structural and dynamical properties of the squirrel Mb have been also deeply investigated by CD, NMR. Furthermore, molecular dynamics studies of Mbs from different species have been performed. In addition, the functional properties of squirrel Mb have been characterized by determining its autoxidation kinetic and thermal stability in comparison with crested porcupine and reindeer Mbs. Interestingly, a higher autoxidation rate was revealed for squirrel Mb with respect to reindeer and crested porcupine Mbs. Even considering the very similar structural fold, molecular dynamics data show a higher conformational mobility of squirrel Mb with respect to reindeer and crested porcupine.

Keywords: Heme-proteins; Homology modeling; Mass spectrometry; Protein purification; Squirrel; Vertebrate globins.