Much of biology is regulated by macromolecular interactions. Understanding the principles that govern the regulation of macromolecular interactions is critical for the rational manipulation of biological processes such as the immune response, gene expression and cellular growth. Allosteric proteins, which alter the interactions between subunits in response to environmental conditions, provide ideal systems for exploring the regulation of macromolecular interactions. We will be using three disparate invertebrate and primitive vertebrate hemoglobins as model systems for investigating atomic-level principles of allosteric protein function. Despite the similar tertiary structure of these hemoglobins, the extent of assembly into cooperative complexes is very different. The goal of this project is to elucidate the structural diversity and common themes that operate to regulate function in this family of proteins. Scapharca dimeric hemoglobin represents the simplest possible model system for cooperative protein function. Our powerful combination of structural, functional and mutational approaches for exploring the cooperative mechanism has revealed a number of important aspects of its function, including the central role of water molecules as sensors of ligation state. We are continuing our approaches to learn how assembly alters ligand pathways and to dissect the coupling between alternate pathways for communication between subunits. A much more complex system is Lumbricus erythrocruorin, which is assembled from 144 hemoglobin subunits and 36 non-hemoglobin (linker) subunits. Our structural results reveal that this molecule is assembled using an intricate hierarchy of symmetry. Our investigation of this molecule is designed to determine the role of two important domains, coiled coils and the cysteine-rich LDL-A module, for formation of this gigantic complex. In addition, we will determine the structural basis for the high cooperativity of this complex. A third system under investigation is the hemoglobin from lamprey, a primitive vertebrate. Unlike other hemoglobins, this hemoglobin gains cooperativity as a result of the concentration dependent equilibrium between different oligomeric states. Our structural analysis suggests a number of hypotheses for the regulation of function, which we will be testing by mutagenesis, kinetic and structural experiments.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Sechi, Salvatore
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University of Massachusetts Medical School Worcester
Other Basic Sciences
Schools of Medicine
United States
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Royer Jr, William E; Omartian, Michael N; Knapp, James E (2007) Low resolution crystal structure of Arenicola erythrocruorin: influence of coiled coils on the architecture of a megadalton respiratory protein. J Mol Biol 365:226-36
Nienhaus, Karin; Knapp, James E; Palladino, Pasquale et al. (2007) Ligand migration and binding in the dimeric hemoglobin of Scapharca inaequivalvis. Biochemistry 46:14018-31
Nichols, Jeffry C; Royer Jr, William E; Gibson, Quentin H (2006) An optical signal correlated with the allosteric transition in Scapharca inaequivalvis HbI. Biochemistry 45:15748-55
Knapp, James E; Pahl, Reinhard; Srajer, Vukica et al. (2006) Allosteric action in real time: time-resolved crystallographic studies of a cooperative dimeric hemoglobin. Proc Natl Acad Sci U S A 103:7649-54
Royer Jr, William E; Sharma, Hitesh; Strand, Kristen et al. (2006) Lumbricus erythrocruorin at 3.5 A resolution: architecture of a megadalton respiratory complex. Structure 14:1167-77
Knapp, James E; Bonham, Michele A; Gibson, Quentin H et al. (2005) Residue F4 plays a key role in modulating oxygen affinity and cooperativity in Scapharca dimeric hemoglobin. Biochemistry 44:14419-30
Royer Jr, William E; Zhu, Hao; Gorr, Thomas A et al. (2005) Allosteric hemoglobin assembly: diversity and similarity. J Biol Chem 280:27477-80
Flores, Jason F; Fisher, Charles R; Carney, Susan L et al. (2005) Sulfide binding is mediated by zinc ions discovered in the crystal structure of a hydrothermal vent tubeworm hemoglobin. Proc Natl Acad Sci U S A 102:2713-8
Strand, Kristen; Knapp, James E; Bhyravbhatla, Balaji et al. (2004) Crystal structure of the hemoglobin dodecamer from Lumbricus erythrocruorin: allosteric core of giant annelid respiratory complexes. J Mol Biol 344:119-34
Knapp, James E; Srajer, Vukica; Pahl, Reinhard et al. (2004) Immobilization of Scapharca HbI crystals improves data quality in time-resolved crystallographic experiments. Micron 35:107-8

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