The goal of this project is to elucidate atomic level principles by which cooperative protein function depends upon the stereochemical details of assembly. We will use three disparate hemoglobins that provide excellent systems for revealing such principles. In each case, the component subunits are folded similarly, but the extent of subunit assembly into cooperative complexes is very different. Nonetheless, there is evidence that aspects of the intersubunit interactions could be similar in all three systems. We are investigating details of the linkage between assembly and ligand binding in these three systems to learn how multiple modes of regulation are achieved within a single protein fold. We are pursuing a powerful combination of structural, functional, and mutational approaches to explore the mechanism of cooperativity in the simple Scapharca dimeric hemoglobin. A major recent finding is that ordered water molecules in the subunit interface can play a direct role in communication between subunits. High resolution crystal structures revealed that a very well ordered cluster of water molecules in the deoxy interface is disrupted upon ligand binding. Mutagenesis and osmotic experiments demonstrate that these water molecules are critical for maintenance of the low affinity conformation. This strongly suggests a mechanism for cooperativity in which the integrity of the water cluster is used as a sensor for ligation state of each subunit. We are exploring the pathway for communication that starts at the heme iron atom and passes through this water network to impact upon the second subunit. The same subunit fold found in Scapharca dimeric hemoglobin is also found in the hemoglobins of Lumbricus and lamprey, but subunit assembly is quite distinct. Nearly two-hundred subunits assemble to form Lumbricus hemoglobin, which shows both strong cooperativity and regulatory features. In contrast, lamprey hemoglobin is monomeric in the oxygenated state, but attains cooperativity from assembly into a lower affinity complex upon oxygen release and proton uptake. We are using x-ray crystallographic analysis to determine the structural basis for allostery and assembly of these molecules. The central role of cooperativity in biological function suggests that principles obtained from this study of three different, but related, allosteric proteins will have wide ranging applications to other physiologically important systems.

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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Laughlin, Maren R
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University of Massachusetts Medical School Worcester
Other Basic Sciences
Schools of Medicine
United States
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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
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
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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