A Phylogenetic and Structural Study of Truncated Hemoglobins

Open Access
Vuletich, David Andrew
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
August 01, 2007
Committee Members:
  • Juliette Lecomte, Committee Chair
  • Philip C. Bevilacqua, Committee Member
  • Michael Thomas Green, Committee Member
  • Donald Bryant, Committee Member
  • Lee Kump, Committee Member
  • truncated hemoglobins
  • NMR
  • H/D exchange
  • Phylogeny
  • heme-protein crosslinking
The hemoglobin superfamily consists of proteins characterized by an ?-helical fold that binds heme (iron protoporphyrin IX) through histidine coordination to the iron. The first characterized members of the superfamily, hemoglobin and myoglobin, function in the respiratory systems of higher organisms for oxygen transport and storage. However, many more members of the superfamily have been discovered in recent years. These genes are found in all the major branches of life, show high levels of sequence divergence, and have adapted to perform different functions. The study of highly adaptable genes gives insight into process of molecular evolution unlike that obtained from the study of stable genes. In addition, the different adaptations of globins may provide insight into the history of oxygen utilization or organisms’ adaptation to increasing oxygen levels. This thesis examines the evolutionary history and biophysical properties of the truncated globins. Because these proteins represent an ancient lineage, their study provides unique insight into how the family originated and what the effects billions of years of evolution are on such an adaptable protein. In addition, the most important capabilities of these globins (ligation, hydrogen bond networks, and ligand stabilization) are defined by a small number of amino acids in the heme pocket. This allows for profound statements to be made through analysis of their variability within different genomes, and the structural repercussions of that variability. Truncated globins (trHbs) constitute one of the three hemoglobin lineages. TrHbs are found in bacteria, plants, and unicellular eukaryotes. They are distantly related to vertebrate hemoglobins and their globin domains are typically shorter than these by 20 to 40 residues. The multiple amino acid deletions, insertions, and replacements result in distinctive alterations of the canonical globin fold and a range of chemical properties. An early phylogenetic analysis categorized trHbs in three groups, I (trHbN), II (trHbO), and III (trHbP). This analysis has been revisited with 111 trHb sequences. It is found that trHbs are orthologous within each group and paralogous across the groups. Group I globins form the most disparate set and separate into two divergent classes. Group II is comparatively homogeneous, whereas Group III displays the highest level of overall conservation. In Group I and Group II globins, an improved description of probable protein-ligand interactions is achieved. Other conservation trends are either confirmed (essential glycines in loops), refined (lining of ligand access tunnel), or newly identified (helix start signal). The Group III globins exhibit recognizable heme cavity residues while lacking some of the residues thought to be important to the trHb fold. An analysis of the phylogenetic trees of each group provides a plausible scenario for the emergence of trHbs, by which the Group II globin gene was the original gene, and the Group I and Group III globin genes were obtained via duplication and transfer events. The Group I trHb from the cyanobacterium Synechocystis sp. PCC 6803 (S6803 Hb) exhibits structural features uncommon to trHbs in that it is endogenously hexacoordinate in the resting state and can covalently modify the heme cofactor. The phylogenetic analysis shows that the positions responsible for these features (HisE7 as the second axial ligand to the iron and HisH16 as the residue forming a covalent bond with the heme 2-vinyl) are both conserved in only one other sequence, the Group I 2/2 globin from the cyanobacterium Synechoccocus sp. PCC 7002 (S7002 Hb). In order to understand better the occurrence of hexacoordination and crosslinking in these globins, S7002 Hb was characterized. Both features were found to be present in S7002 Hb, and comparisons of the biophysical data show they are manifested in similar ways. However, the characteristics of ligand binding were found to differ between the two proteins. Recombinant S7002 Hb (rHb) can be readily prepared as an apoglobin (apo-rHb), a noncross-linked hemichrome (ferric iron and two histidine axial ligands, rHb-R), and a cross-linked hemichrome (rHb-A). To determine the effects of heme binding and subsequent cross-linking, apo-rHb, rHb-R, and rHb-A were subjected to thermal denaturation and 1H/2H exchange experiments. Interpretation of the latter data was based on NMR assignments obtained with uniformly 15N- and 13C,15N-labeled protein. Apo-rHb was found to contain a cooperative structural core, which was extended and stabilized by heme binding. Crosslinking resulted in further stabilization attributed mainly to an unfolded state effect. Protection factors, i.e., the degree of hydrogen exchange retardation due to organized structure, were higher at the cross-link site and near His70 in rHb-A than rHb-R. In contrast, other regions became less resistant to exchange in rHb-A. These included portions of the B helix and the E helix, which undergo large conformational changes upon exogenous ligand binding to the iron. Thus, the crosslink readjusted the dynamic properties of the heme pocket. 1H/2H exchange data also revealed that the B, G, and H helices formed a robust core regardless of the presence of the heme or the crosslink. This motif likely encompasses the early folding nucleus of trHbs. The solution structure of S7002 rHb-A was solved and compared to other trHbs, myoglobin, and the apo and noncrosslinked S7002 rHb. It was found that S7002 and S6803 rHb-As have similar secondary and tertiary structure, including a kink in the H helix. This kink places the C terminal end of the H helix near the heme, a position facilitating the formation of the crosslink. The helix kink is observed in the structures of all other Group I trHbs, regardless of the ability of residue H16 to react with the heme. Comparisons of these structures identified a conserved heme-valine contact not present in vertebrate globins. S7002 and S6803 rHb-A differed in the EF loop structure, a detail that may be responsible for the distinct rates of S7002 and S6803 rHb cyanide binding. Finally, chemical shift data were used to compare the secondary structure of S7002 rHb-R and apo-rHb to that of rHb-A. It was found that all three forms of the protein share similar helical regions. These data were used with the S7002 apo-rHb chemical denaturation data to assess the extent of refolding induced by association with the heme group.