Understanding a Understanding a Millenium Millenium of of Hemoglobin Evolution: Correlating Hemoglobin Evolution: Correlating

Conformation with BindingConformation with Binding

Wendell P. Griffith; and Igor A. KaltashovUniversity of Massachusetts, Amherst, MA

OverviewOverview

• Previous work showed that the α- and β- chains played asymmetric roles in the assembly of bovine hemoglobin (Hb). The binding of a partially unstructured apo-β-chain to a tightly folded holo-α-chain to form a heme-deficientdimer is the initial step of hemoglobin assembly.

• This phenomenon was found in all other mammalian hemoglobins analysed.

• In fish hemoglobin the roles of the α- and β- chains are reversed. This suggests that the asymmetric behavior evolved separately within each class of organism.

IntroductionIntroduction• Hemoglobins are O2 transport proteins.

• They are universally present in higher organisms as tetramers, and are also found in some bacteria and plants as monomers and may be as large as the 144 subunit hemoglobins of annelids.

• Remarkable diversity in 4° assemblage is observed among invertebrate and primitive vertebrate Hbs.

• In bovine hemoglobin there is a highly asymmetric behavior between the α- and β-chain conformations.

• This phenomenon may have arisen due to evolutionary pressure upon Hb encapsulation in red blood cells.

Evolution of theEvolution of the GlobinGlobin SpeciesSpecies• The Hb molecule 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 hemoglobin molecule with separate α and β chains occurred 500 – 600 M years later.

• Most studies of globin evolution have been by the comparison of amino acid sequences (1° structure) for myoglobins and hemoglobins from various species.

• We study evolution by looking at the changes it has caused in protein conformational flexibility during the hemoglobin assembly process (formation of 4° structure).

GlobinGlobin Evolutionary TreeEvolutionary Tree• The tree was

constructed by plotting relative differences between amino acid sequences.

• The more closely related species appear on closer branches of the tree.

• The tree’s branching closely mirrors the evolutionary “Tree of Life”.

ExperimentalExperimental

• Mass Spectrometry: JMS-700 MStation (JEOL, Tokyo, Japan) two-sector instrument with standard ESI source

• Materials:– Bovine hemoglobin (Sigma # H2500)– Human hemoglobin (Sigma # H7379)– Baboon hemoglobin (Sigma # H2010)– Porcine hemoglobin (Sigma # H4131)– Atlantic cod hemolysate (donation from UMASS Marine

Station)

Mammalian Mammalian HemoglobinsHemoglobins+

↑↓

↑↓

↑↓

Comparison with Isolated ChainsComparison with Isolated Chains(α*β*)+12

(α*β*)2+17

(α*)+9

β+16

(α*β)+13

(α*)+8

β+9β+8 (β*)+8

(β*β*)+12

Hb Hb H vs. Normal Hemoglobin AH vs. Normal Hemoglobin A

Normal Hemoglobin (Hb A)Alpha Thalassemic Hemoglobin (Hb H)

Atlantic Cod HemoglobinAtlantic Cod Hemoglobin(α*β*)2

+17

α+19

(αβ*)+12

(β*)+8

+

↑↓

↑↓

↑↓

ConclusionsConclusions• The asymmetry between globin chains is not surprising.

– We believe that it arose due to evolutionary pressure as a vital safe-guard to ensure the correct assembly of the α2β2tetramer.

• The roles of the α- and β-chains in fish Hbs are reversed.– This suggests that the asymmetry in conformation arose

independently in the fish and mammalian systems, in parallel. This is evident when considering that fish and placental mammals are on different branches of the evolutionary tree.

Future WorkFuture Work

• Sample hemoglobins from organisms on other branches of the evolutionary tree.– Does the asymmetry in conformation exist in all

tetrameric hemoglobins?– Is there any asymmetry in conformation in the chains

of dimeric and the much larger extracellular Hbs?

• Examine the behavior of isolated Hb chains– Can the asymmetric behavior be characterized?

AcknowledgementsAcknowledgements

• Stephen J. Eyles

• Prof. Herbert O. Hultin– UMass Marine Station

• University of Massachusetts, Department of Chemistry

• Funded by NIH grant R01 GM61666

ReferencesReferencesGriffith, W. P., and Kaltashov, I. A. (2003) Biochemistry42(33), 10024 - 10033.

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Perutz, M. (1997) Science is not a quiet life:Unravelling the atomic mechanism of haemoglobin, World Scientific Publishing Co. Pte. Ltd., Singapore.

Vasudevan, G., and McDonald, M. J. (1997) J. Biol. Chem. 272, 517-524.

Vasudevan, G., and McDonald, M. J. (2002) Curr. Protein Pept. Sci. 3, 461-466.