Abstract

The aim of this project is the characterization of the functional properties of variants of human hemoglobin by examination of the equilibria and kinetics of their reactions with ligands. The purpose of this undertake is to contribute to the further development and testing of predictive stereochemical model which has been developed by this Program Project. Hemoglobin variants, as well as hemoglobins containing two different alphabeta dimers (mixed-mutant hybrid hemoglobins) and hemoglobins containing different metalloporphyrins (mixed-metal hybrids), will be prepared by Core C, using site directed mutagenesis and expression of globin chains in E. coli., followed by isolation and assembly. Equilibria of oxygen binding in solution to the mutant hemoglobins will be measured. Kinetic studies will be carried out using stopped-flow and flash photolysis techniques to examine the effects of mutations on the properties of the R and T quaternary states of the hemoglobin molecule, the relative stabilities of these states and the stability of the tetramer with respect to dissociation into alphabeta dimers. Properties of the T states of these materials will also be examined by measuring the equilibria of oxygen binding to T state crystals grown in polyethylene glycol as well as to T state molecules embedded in silica gels. Properties of the alpha and beta chains of hemoglobin variants within the T quaternary state, and the functional properties of partially liganded hemoglobin variants will be assessed by examining the equilibrium and kinetic properties of symmetric and asymmetric mixed-metal and mixed-mutant hybrid hemoglobins.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM058890-02
Application #
6301803
Study Section
Project Start
2000-01-01
Project End
2000-12-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
2
Fiscal Year
2000
Total Cost
$209,639
Indirect Cost

  Institution

Name
University of Iowa
Department
Type
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242

  Publications

Kwiatkowski, Laura D; Hui, Hilda L; Karasik, Ellen et al. (2007) Mutations of the betaN102 residue of HbA not only inhibit the ligand-linked T to Re state transition, but also profoundly affect the properties of the T state itself. Biochemistry 46:2037-49
Das, Tapan K; Dewilde, Sylvia; Friedman, Joel M et al. (2006) Multiple active site conformers in the carbon monoxide complexes of trematode hemoglobins. J Biol Chem 281:11471-9
Samuni, Uri; Roche, Camille J; Dantsker, David et al. (2006) Modulation of reactivity and conformation within the T-quaternary state of human hemoglobin: the combined use of mutagenesis and sol-gel encapsulation. Biochemistry 45:2820-35
Dantsker, David; Roche, Camille; Samuni, Uri et al. (2005) The position 68(E11) side chain in myoglobin regulates ligand capture, bond formation with heme iron, and internal movement into the xenon cavities. J Biol Chem 280:38740-55
Kavanaugh, Jeffrey S; Rogers, Paul H; Arnone, Arthur et al. (2005) Intersubunit interactions associated with Tyr42 alpha stabilize the quaternary-T tetramer but are not major quaternary constraints in deoxyhemoglobin. Biochemistry 44:3806-20
Kavanaugh, Jeffrey S; Rogers, Paul H; Arnone, Arthur (2005) Crystallographic evidence for a new ensemble of ligand-induced allosteric transitions in hemoglobin: the T-to-T(high) quaternary transitions. Biochemistry 44:6101-21
Viappiani, Cristiano; Bettati, Stefano; Bruno, Stefano et al. (2004) New insights into allosteric mechanisms from trapping unstable protein conformations in silica gels. Proc Natl Acad Sci U S A 101:14414-9
Samuni, Uri; Dantsker, David; Juszczak, Laura J et al. (2004) Spectroscopic and functional characterization of T state hemoglobin conformations encapsulated in silica gels. Biochemistry 43:13674-82
Peterson, Eric S; Shinder, Roman; Khan, Imran et al. (2004) Domain-specific effector interactions within the central cavity of human adult hemoglobin in solution and in porous sol-gel matrices: evidence for long-range communication pathways. Biochemistry 43:4832-43
Hui, Hilda L; Kwiatkowski, Laura D; Karasik, Ellen et al. (2004) Ligand binding to symmetrical FeZn hybrids of variants of human HbA with modifications in the alpha1-beta2 interface. Biochemistry 43:7843-50

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