Globins are small globular metalloproteins that contain heme prosthetic groups and have a wide range of biological functions. Recent advancements in sequencing technology allowed for the recognition of novel six-coordinated vertebrate globins. Although these proteins share high structural similarities with pentacoordinate proteins, the presence of the hexacoordination, as well as an intracellular disulfide bridge, make hexacoordinate globins an excellent model for understanding structural determinants that influence protein stability. Previous studies in our group revealed an increased pH stability of hexacoordinate GbX. To provide a further understanding of structural features that contribute to the increased stability of this protein, we combine “in solution” and “in gas” approaches to characterize unfolding mechanism of GbXWT, GbXC65A, and GbXH90V. GbXC65A has similar stability as the wild-type protein towards unfolding by GuHCl with ΔG = 13 kcal mol-1 and 15.4 kcal mol-1 respectively. However, the stability of GbXH90V is significantly reduced, ΔG = 4.8 kcal mol-1. Emission data show that unfolding of GbXC65A is more complex as an unfolding intermediate has been detected at [GuHCl] = 5.0-5.5 M. The TIMS-MS technique also confirmed the high stability of GbX and GbXC65A. GbX is significantly more stable than pentacoordinate myoglobin, (ΔG = 5.0 kcal mol-1) and hexacoordinate Ngb, (ΔG = 7.3 kcal mol-1), suggesting that other factors than the presence of coordination bond between distal histidine and heme iron contribute to the stability of this protein. The comparison of the amino acid sequence of Mb, Ngb, Cygb, and GbX reveals a smaller percentage of nonpolar amino acid residues and an increased amount of charged amino acid residues in GbX. A higher ratio of charged amino acid residues may result in unusual GbX stability which was previously observed in the study of hydrogen bond’s contribution from the side chain residues to protein stability.

Structural characterization of folding of GbX with focus on intrinsic parameters influence protein stability

ACS Fall 2021

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