Fundamentals of Biochemistry Third Edition Donald Voet • Judith G. Voet • Charlotte W. Pratt Chapter 7 Protein Function: Myoglobin and Hemoglobin, Muscle Contraction, and Antibodies Copyright © 2008 by John Wiley & Sons, Inc.

Myoglobin 153 residues with 8 alpha helices (lettered), double letters indicate interhelix sections.

The heme ring binds oxygen at the iron’s axial position The heme ring binds oxygen at the iron’s axial position. The other axial position is stabilized by ion-dipole interaction with a histidine side chain

Stabilization of the heme complex His F8 (seen in the previous slide) stabilizes the iron (II) ion from “below”; when O2 is present, His E7 stabilizes it. There is also a color change from purple to scarlet as the oxygen binds to the heme ring; however, the iron (II) never oxidizes fully to iron (III).

Oxygen saturation curve for myoglobin p50 is defined as the oxygen pressure at which the oxygen carrier is half-saturated

There are other oxygen carriers: hemocyanin, for instance. The “active site” contains two copper (II) ions, each stabilized by three His side chains

Relaxed (R – in blue) and Tense (T – in red) conformations of hemoglobin Hemoglobin is an α2β2 tetramer (alternate subunits shown by shading), held together mostly by interchain hydrophobic interactions with some hydrogen bonds and ion pairs

Deoxyhemoglobin Wide central channel

Hemoglobin A 15° rotation of one of the αβ units narrows central channel

Oxygen saturation curves for myoglobin and hemoglobin Note the sigmoidal (S curve) shape of the hemoglobin curve, which is characteristic of a protein that exhibits cooperative behavior Allosteric effect: when the binding of one ligand on a protein affects the binding of another ligand elsewhere on the protein

Hill plot shows cooperativity of hemoglobin Archibald Hill (1911) Hill equation Slope of 1 (n=1) means noncooperativity

The mechanism of hemoglobin conformational change But how can a hemoglobin molecule “know” that one of its subunits has bound an oxygen already? Perutz, M.F., “Structure of hemoglobin” Brookhaven Symp Biol. 1960 Nov;13:165-83

The mechanism of hemoglobin conformational change But how can a hemoglobin molecule “know” that one of its subunits has bound an oxygen already? When an oxygen binds to the heme, the heme ring “bends” such that it is planar; this in turn pulls His F8 down 0.6 Å

The mechanism of hemoglobin conformational change But how can a hemoglobin molecule “know” that one of its subunits has bound an oxygen already? When an oxygen binds to the heme, the heme ring “bends” such that it is planar; this in turn pulls His F8 down 0.6 Å Which then moves helix F 1 Å

The mechanism of hemoglobin conformational change Those changes cause changes in the α1β2 and α2β1 interactions. For the α1β2 interaction shown, the Asp-Tyr connection is broken, and an Asn-Asp connection is made

The mechanism of hemoglobin conformational change Finally, the ion pairs at the C and N termini of the different subunits shift to accommodate the new conformation, and the change is complete!

Oxygen saturation curve for hemoglobin at varying pH Bohr effect: increasing pH removes protons from hemoglobin, stimulating hemoglobin to bind more O2

Hemoglobin and myoglobin in oxygen and carbon dioxide transport

Oxygen saturation curve for hemoglobin with different allosteric effectors (modulators) 2,3–BPG is derived directly from glycolysis Does BPG make hemoglobin more or less cooperative?

BPG binds in the central cavity of deoxyhemoglobin to the two β units’ N-terminal amino groups. Therefore, BPG stabilizes the T conformation and decreases hemoglobin’s oxygen affinity. When the T  R shift occurs, the BPG is expelled as the cavity narrows.

Adaptation to high altitudes – when confronted with a much lower environmental pO2 , the body responds by producing a high concentration of BPG, shifting the curve to the right, allowing more oxygen to be released than without the BPG.

Two models of allosteric behavior: the symmetry model All subunits make the T  R shift simultaneously, and this can happen at any point with any number of ligands attached

Two models of allosteric behavior: the sequential model The T  R shift occurs to each subunit as it binds the ligand. Amusingly, hemoglobin does both – the large quaternary shift that preserves the overall symmetry of the molecule accompanied by small tertiary structure shifts as each oxygen binds.

Sickle-cell variant of hemoglobin – a one-residue change in the primary sequence of each β chain: Val (6) Glu – enough to change the shape of the erythrocyte into a linear structure, not a disk.

The mutant valine residue can now interact with two nonpolar residues on a different chain of a different molecule – something glutamic acid could not do.

The mutant valine residue can now interact with two nonpolar residues on a different chain of a different molecule – something glutamic acid could not do. And make these linear structures – hemoglobin S fibers

The S fibers dissolve when oxygenated

The S fibers dissolve when oxygenated Hydroxyurea increases the amount of fetal hemoglobin, which allows for oxygen transport

The protozoan that causes malaria must live in erythrocytes for some of its life; without exclusively normal-shaped blood cells, the sickle-cell carrier suffers less from malaria.

Muscle contraction is another example of allosteric conformational change: the myosin protein binds ATP (adenosine triphosphate) and the actin protein

Antibodies are proteins produced by B cells (in the bone marrow), desgined to bind to specific molecules (antigens)

Antibodies are also known as immunoglobulins (Ig); they have the general structure α2β2, where α is the “heavy” (higher mol. wt.) chain and β is the “light” chain

Another way to consider the structure of antibodies is to digest the molecule with the enzyme papain, which splits the antibody into three fragments: two Fab, which contain the antigen-binding site and one Fc. The Fab site has the variable region which is the site of antigen recognition, so therefore this region varies in sequence from antibody to antibody. Once the antigen binds, the antibody changes shape and triggers an immune response.

Monoclonal antibodies