Abstract
The structure of alpha-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free alpha-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three alpha-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to alpha-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer alpha1beta1 interface in tetrameric HbA.
Highlights
The structure of ␣-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free ␣-hemoglobin, has been determined using NMR spectroscopy
Both the wild-type AHSP and the P30A mutant bind to ␣-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form
Conformational Heterogeneity of AHSP—The line width and dispersion of the signals observed in the 1H-15N HSQC spec
Summary
DNA Cloning—The DNA sequence for the full-length human AHSP was cloned from a human fetal liver cDNA library (Clontech) using the appropriate 5Ј- and 3Ј-end primers, which contained BamHI and EcoRI restriction sites, respectively. The resulting PCR product was ligated into a modified pRSETA vector (Invitrogen) containing a soluble hexahistidine-tagged GroEL fusion protein. Protein Expression and Purification—The plasmid was transformed into C41 host cells [5] and grown in 2ϫ TY medium or a modified K-MOPS minimal medium [6] with 20 mM 15NH4Cl and 0.4% [13C]glucose containing 50 g/ml ampicillin. The cells were grown at 37 °C, This paper is available on line at http://www.jbc.org.
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