Several novel members of the vertebrate globin family were recently discovered with unique structural features that are not found in traditional penta-coordinate globins. Here we combine structural tools to better understand and recognize molecular determinants that contribute to the stability of hexacoordinate globin X (GbX) from Danio rerio (zebrafish). pH-induced unfolding data indicates increased stability of GbX with pHmid of 1.9 ± 0.1 for met GbXWT, 2.4 ± 0.1 for met GbXC65A, and 3.4 ± 0.1 for GbXH90V. These results are in good agreement with GbX unfolding experiments using GuHCl, where a ΔGunf 13.8 ± 2.5 kcal mol-1 and 16.3 ± 2.6 kcal mol-1 are observed for metGbXWT, and metGbXC65A constructs, respectively, and diminished stability is measured for GbXH90V, ΔGunf = 9.5 ± 3.6 kcal mol-1. The metGbXWT and metGbXC65A also exhibit high thermal stability (melting points of 118 °C and 107 °C, respectively). Native ion mobility - mass spectrometry (IM-MS) experiments showed a narrow charge state distribution (9-12+) characteristics of a native, structured protein; a single mobility band was observed for the native states. Collision induced unfolding IM-MS experiments showed a two-state transition, in good agreement with the solution studies. GbXWT retains the heme over a wide range of charge states, suggesting strong interactions between the prosthetic group and the apoprotein. The above results indicate that in addition to the disulfide bond and the heme iron hexa-coordination, other structural determinants enhance stability of this protein.
Keywords: Collision induced unfolding; Folding; Globin X; Heme protein; Hexacoordination; Stability.
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