Role of Heme in the Folding and Assembly of Globins

[摘要] Globins constitute a superfamily of proteins that bind a heme cofactor and havediverse physiological roles, ranging from oxygen management to nitric oxide scavengingto gas sensing. Sperm whale myoglobin (Mb) has remained the key system for a widevariety of biophysical investigations of globin function and structure. It possesses theconserved and well-characterized 3-on-3 helical fold, which produces a hydrophobicpocket for heme binding and exogenous ligand coordination. Upon removal of the hemeprosthetic group, the protein loses -40% of its native secondary structure but still servesas a model apo-protein with high helical content for unfolding studies. At least one onpathwayapoMb intermediate is populated kinetically during folding or at eqUilibriumduring acid, urea, or GuHCl-induced denaturation. In contrast, much less is known aboutthe folding and assembly of the holoMb, and how heme confers resistance todenaturation. It is presumed that in vivo, oxidation of the heme iron from the ferrous tothe ferric state precedes denaturation; however the detailed mechanism for the unfoldingof the ferric holoprotein has remained enigmatic. The work presented defines the role ofheme quantatively in the folding and assembly of Mb and possibly for the assembly ofglobins in general. We have demonstrated that ferric holoMb unfolds via a hemin-boundintermediate state, which has the characteristics of a reversible hemichrome species thatis also on the pathway to assembly back to the holoprotein. Similar hemichromeintermediates are observed during unfolding of adult human HbA. The formation of thishemichrome state is very relevant to the biology of erythropoiesis, hemoglobindegradation and the formation of Heinz bodies in circulating red blood cells. We havealso tested our methodology using a simpler monomeric hemoglobin from Cerebratuluslacteus that possesses low stability and does not populate an intermediate capable ofbinding heme. All of these results have general implications in biophysics for howcofactor-containing proteins fold, in biology for the understanding of erythropoiesis (i.e.,in vivo hemoglobin assembly), and in biotechnology for the understanding of how tooptimize heterologous expression yields of recombinant holo heme proteins in E. coli foreither research or commercial purposes.

[发布机构] Rice University

[效力级别] Cellular