Role of Heme in the Folding and Assembly of Globins

• Main Author: Culbertson, David Sean
• Other Authors: Olson, John S.
• Format: Thesis
• Language: English
• Published: 2010
• Subjects: Biochemistry; Cellular biology; Sperm whale
• Online Access: https://hdl.handle.net/1911/64410

Globins constitute a superfamily of proteins that bind a heme cofactor and have diverse physiological roles, ranging from oxygen management to nitric oxide scavenging to gas sensing. Sperm whale myoglobin (Mb) has remained the key system for a wide variety of biophysical investigations of globin function and structure. It possesses the conserved and well-characterized 3-on-3 helical fold, which produces a hydrophobic pocket for heme binding and exogenous ligand coordination. Upon removal of the heme prosthetic group, the protein loses -40% of its native secondary structure but still serves as a model apo-protein with high helical content for unfolding studies. At least one onpathway apoMb intermediate is populated kinetically during folding or at eqUilibrium during acid, urea, or GuHCl-induced denaturation. In contrast, much less is known about the folding and assembly of the holoMb, and how heme confers resistance to denaturation. It is presumed that in vivo, oxidation of the heme iron from the ferrous to the ferric state precedes denaturation; however the detailed mechanism for the unfolding of the ferric holoprotein has remained enigmatic. The work presented defines the role of heme quantatively in the folding and assembly of Mb and possibly for the assembly of globins in general. We have demonstrated that ferric holoMb unfolds via a hemin-bound intermediate state, which has the characteristics of a reversible hemichrome species that is also on the pathway to assembly back to the holoprotein. Similar hemichrome intermediates are observed during unfolding of adult human HbA. The formation of this hemichrome state is very relevant to the biology of erythropoiesis, hemoglobin degradation and the formation of Heinz bodies in circulating red blood cells. We have also tested our methodology using a simpler monomeric hemoglobin from Cerebratulus lacteus that possesses low stability and does not populate an intermediate capable of binding heme. All of these results have general implications in biophysics for how ...