Download Understanding a Millennium of Hemoglobin Evolution: Correlating

Understanding a Millennium of Hemoglobin Evolution:
Correlating Conformation with Binding
Wendell P. Griffith; and Igor A. Kaltashov
Department of Chemistry, University of Massachusetts, Amherst, MA 01003
Hemoglobins are dioxygen transport proteins. They are universally present in higher vertebrates as a
tetrameric protein, and are found in some bacteria and plants as monomers. Remarkable diversity in
quaternary (4°) assemblage is observed among invertebrates and primitive vertebrate hemoglobins.
These range from the 144-subunit earthworm hemoglobins to dimeric molluscan hemoglobins, and the
monomeric leghemoglobins of some plants. Previous work has shown that the behaviors of the alpha
and beta chains of bovine are highly asymmetric.1 They both adopt different conformations as a
prerequisite for binding and assembly. Current work uses ESI MS to study the conformational flexibility
and binding (assembly) of various hemoglobins. This is done in an attempt to shed light on the
differences in hemoglobin structure between species and possible causes for these differences due to
evolutionary pressure. It also compares the behavior of the isolated chains to similar chains within the
intact hemoglobin complex.
The hemoglobin (Hb) molecule, being one of the most well-studied protein systems, has a long
evolutionary history. Its discovery in virtually all kingdoms of organisms has shown that the ancestral
gene for Hb is ancient. It has been used to date the separation of the vertebrates and invertebrates some
1000 million (M) years ago. The emergence of a vertebrate Hb molecule with separate α- and β- chains
occurred 500 – 600 M years later. Most studies of globin evolution to date have been by the comparison
of amino acid sequences (1° structure) for myoglobins and hemoglobins from various species and by
comparing their static 4° structures.2 Our approach to studying the evolution if the hemoglobin system is
by looking at the changes it has caused in protein conformational flexibility of either Hb chain during the
assembly process (formation of 4° structure).
All mass spectra were acquired on a JMS-700 MStation (JEOL, Tokyo, Japan) two-sector mass
spectrometer equipped with a standard ESI-source. All hemoglobin solutions were prepared by dilution to
a concentration of approximately 10 µM in pH 8 ammonium acetate solutions between 10 and 100 mM.
All sample solutions were equilibrated at room temperature (24°C) for 1 hour prior to analysis.
Conformational flexibility was detected according to the method developed by Dobo and Kaltashov.3
In all of the vertebrate hemoglobins analyzed, the α- and β- chains play asymmetric roles in the assembly
process. In all of the mammalian (bovine, human, and porcine shown in Figure 1) hemoglobins analyzed,
the α-chains maintain a very highly structured compact conformation as suggested by low protein charge
density and narrow charge-state distribution. They are very competent in heme binding. The β-chains,
on the other hand, in addition to lacking a heme group, exhibit a comparably significantly higher charge
density and a broader charge-state distribution. Isolated β-chains also oligomerize to form tetramers
upon addition of heme to a solution of this protein chain. This is most unlike the isolated α-chains, which
remain monomeric upon heme acquisition. This is important as it demonstrates the formation of the nonfunctional hemoglobin H (HbH), which is found in individuals in an alpha-thalassemic disease state. This
asymmetry in comformational flexibility between the two chains during the assembly processes is quite
remarkable when considering how similar the α- and β- chains are in the HbA (normal human
hemoglobin: PDB ID 2HHB) structure and also how similar the HbA assembled molecule is with HbH
(Figure 2).
In the fish hemoglobin studied (from Gadus morhua, the Atlantic cod), the roles of the α- and β- chains
are reversed (Figure 3).
This suggests that the asymmetric behavior evolved in parallel, but
independently within each class of organism. This is not surprising as placental mammals and modern
fish are from completely different branches of the evolutionary “Tree of Life”. The position of the
branching is immediately following the point of gene duplication where evolution caused there to be
separate genes to code for α- and β- chains.
References:
1. Griffith, W. P., and Kaltashov, I. A. (2003) Biochemistry 42(33), 10024 - 10033.
2. Dickerson, R. E., and Geis, I. (1983) Hemoglobin: structure, function, evolution, and pathology,
Benjamin/Cummings Pub. Co., Menlo Park, Calif.
3. Dobo, A., Kaltashov, I. A. (2001) Anal. Chem. 73(20), 4763-4773.
Hb H vs. Normal Hemoglobin A
Mammalian Hemoglobins
+
↑↓
↑↓
↑↓
Normal Hemoglobin (Hb A)
Alpha Thalassemic Hemoglobin (Hb H)
Figure 2. Overlaid molecules of normal
hemoglobin (HbA, PDB ID 2HHB) and alphaFigure 1.
ESI mass spectra of mammalian
thalassemic hemoglobin (HbH, PDB ID
hemoglobins at near physiological pH.
1CBL) showing similarity in 4° structure.
Atlantic Cod Hemoglobin
(α*β*)2+17
+
↑↓
(αβ*)+12
(β*)+8
↑↓
α+19
↑↓
Figure 3. ESI mass spectra of Gadus morhua (the Atlantic Cod) hemoglobin at
near physiological pH. Note that the asymmetric roles in conformational
flexibility of the α- and β- chains are reversed.