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Foundation in Biomedical Sciences GMS 6001

PROTEIN FUNCTION
Gloria C. Ferreira, Ph.D.
August 30, 2013

OBJECTIVES:
To understand how proteins (e.g., immunoglobins and hemoglobin) function by binding to specific ligands. To recognize the forces that dictate the interaction of proteins with their cognate ligands. To determine the dissociation constant of a ligand to a protein.

To recognize the structure of immunoglobulin G and its antigen binding region.


To recognize the structure of hemoglobin and its allosteric behavior.
READING ASSIGNMENT: Alberts, Johnson, Lewis, Raff, Roberts and Walter (2008), Molecular Biology of the Cell Garland 5th edition, Chapter 3.

KEY POINTS:
Proteins function by binding to ligands in a specific manner. The specificity is dictated by the nature of the amino acid side chains of the protein. The dissociation constant of a protein can be determined using fitting of the data to the equation defining the binding process or Scatchard analysis.

Immunoglobulin G has 2 light and 2 heavy chains. Variable regions of the light and heavy chains are used to form the pocket for binding of antigen.
Hemoglobin is also a tetramer: it is comprised of 2 alpha and 2 beta chains. Hemoglobin binds oxygen in a cooperative manner; it is an allosteric protein.

Proteins Bind to Other Molecules

Fig. 1. The selective binding of a protein to another molecule. Many weak bonds are needed to enable a protein to bind tightly to a second molecule, which is called a ligand for the protein. A ligand must therefore fit precisely into a proteins binding site, so that a large number of noncovalent bonds form between the protein and the ligand. [Adapted from Alberts et al. (2008) in Molecular Biology of the Cell, Chap. 3]. The ability of a protein to bind selectively and with high affinity to a ligand depends on the formation of a set of weak, noncovalent bonds Hydrogen bonds Electrostatic attractions van der Walls attractions Favorable hydrophobic interactions

The Binding Site of a Protein

Fig. 2. Generation of a binding site. The folding of the polypeptide chain typically creates a crevice or cavity on the protein surface. This crevice contains a set of amino acid side chains disposed in such a way that they can form noncovalent bonds only with certain ligands. [Adapted from Alberts et al. (2008) in Molecular Biology of the Cell, Chap. 3].

Fig. 3. The binding site of a protein. The binding site possesses several amino acid side chains that form H bonds and salt links, as well as hydrophobic interactions and van der Waals forces. [Adapted from Alberts et al. (2008) in Molecular Biology of the Cell, Chap. 3].

Active Site of an Enzyme

Fig. 4. An unusually reactive amino acid at the active site of an enzyme. This example is the catalytic triad found in chymotrypsin, elastase and other serine proteases. The aspartic acid side chain (Asp 103) induces the histidine (His 57) to remove the proton from serine 195 (Ser 195). This activates the serine to form a covalent bond with the enzyme substrate. The many convolutions of the polypeptide chain are omitted here. In sum, the side chains may interact with each other to generate highly reactive groups (e.g., 1 alcohols). [Adapted from Alberts et al. (2008) in Molecular Biology of the Cell, Chap. 3].

An Antibody

Fig. 5. An antibody molecule. A. A typical antibody molecule is Y-shaped and has two identical binding sites for its antigen, one on each arm of the Y. The protein is composed of four polypeptide chains (two identical heavy chains and two identical and smaller light chains) held together by disulfide bonds. Each chain is made up of several different immunoglobulin domains, here shaded either blue or gray. The antigen-binding site is formed where a heavy-chain variable domain (VH) and a light-chain variable domain (VL) come close together. There are the domains that differ most in their sequence an structure in different antibodies. Each domain at the end of the two arms of the antibody molecule forms loops that bind to the antigen. Note that the different sequences of amino acids in the variable region of the light and heavy chains are responsible for the different specificities of the IgGs. In B. the finger-like loops (red) contributed by the VL domain. Note that the major type of secondary structure is the antiparallel beta sheet. [Adapted from Alberts et al. (2008) in Molecular Biology of the Cell, Chap. 3].

Non-covalent Bonds between Two Colliding Molecules

Fig. 6. How non-covalent bonds mediate interactions between macromolecules. [From Alberts et al. (2008) in Molecular Biology of the Cell, Chap. 3].

Binding Energies and Equilibrium Constants

Fig. 7. Relating binding energies to the equilibrium constant for an association reaction. A. The equilibrium between molecules A and B and the complex AB is maintained by a balance between the two opposing reactions shown in panels 1 and 2. Molecules A and B must collide if they are to react, and the association rate is therefore proportional to the product of their individual concentrations [A] x [B] (Square brackets indicate concentration.) As shown in panel 3, the ratio of the rate constants for the association and the dissociation reactions is equal to the equilibrium constant (K) for the reaction. [From Alberts et al. (2008) in Molecular Biology of the Cell, Chap. 3].

Binding Energies and Equilibrium Constants

Fig. 8. Relating binding energies to the equilibrium constant for an association reaction. The equilibrium constant in panel 3 of Fig. 7 (previous slide) is that for the reaction A + B AB, and the larger its value, the stronger the binding between molecules A and B. Note that for every 1.41 kcal/mole (5.91 kJ/mole) decrease in free energy the equilibrium increase by a factor of 10 at 37 oC. The equilibrium constant here has units of liters/mole: for simple binding interactions it is also called the association constant, denoted Ka. The reciprocal of Ka is called the dissociation constant, Kd (in units of moles/liter). [From Alberts et al. (2008) in Molecular Biology of the Cell, Chap. 3].

Binding Energies and Equilibrium Constants

The effect of Keq on binding of two molecules together.

Fig. 9. Small changes in the number of weak bonds can have drastic effects on a binding interaction. This example illustrates the dramatic effect of the presence or absence of a few noncovalent bonds in a biological context. [Adapted from Alberts et al. (2008) in Molecular Biology of the Cell, Chap. 3].

Determination of Dissociation Constant

[B]/[F] = - [B]/Kd+ [Rt]/Kd

Fig. 10. Determination of dissociation constant (Kd) of a ligand from a protein. The Kd value can be determined from the fitting of the data to the equation A = (Amax[L])/(Kd+[L]) OR using Scatchard analysis ([B]/[F] = - [B]/Kd+ [Rt]/Kd)

Myoglobin and Hemoglobin

Fig. 11. Three-dimensional structure of myoglobin. A schematic view shows that the protein consists largely of a-helices. The heme group is shown in black and the iron atom is shown as a purple sphere.

Fig. 12. The a2b2 tetramer of human hemoglobin. The structure of the two identical a subunits (red) is similar to, but not identical with, that of the two identical b subunits (yellow). The molecule contains 4 heme groups.

Myoglobin and Hemoglobin

Fig. 13. Myoglobin is a monomer, while hemoglobin is a heterotetramer (22).

Hemoglobin

Fig. 2. Cooperativity enhances oxygen delivery by hemoglobin.

Torr a unit of pressure equal to that exerted by a column of mercury 1 mm high at 0oC and standard gravity (1 mm Hg).

Hemoglobin and Allosteric Regulation

K1

K2

L
L

K3

L
L L

K4

L L

L L

Fig. 3. Simple sequential model for a tetrameric allosteric enzyme (or tetrameric hemoglobin). The binding of a ligand (L) to a subunit changes the conformation of that particular subunit from the T (square) to the R (circle) form. This transition affects the affinity of the other subunits for the ligand.

Allostery is also a property of the O2-binding protein hemoglobin. The binding of O2 to hemoglobin isolated from red blood cells displays sigmoidal behavior, which is indicative of cooperation between subunits (Fig. 2). What is the physiological significance of the cooperative binding of O2 by hemoglobin?

Hemoglobin

Myo

Fig. 14. Cooperativity enhances oxygen delivery by hemoglobin. Hemoglobin, which exhibits cooperative binding of oxygen, releases more O2 to the tissues than a protein that does not possess cooperative binding. (pO2, partial pressure of oxygen) [From Berg, Tymoczko and Stryer (2002) 5th Edition]

Oxygen Binding to Hemoglobin

Fig. 17. Oxygen binding initiates structural changes. The iron ion moves into the plane of the heme on oxygenation. The proximal histidine is pulled along with the iron ion. Binding of O2 to the heme reduces the radius of the Fe2+ and pulls the proximal His towards the planar ring, causing a conformational change throughout the subunit.

Hill Equation

Hill equation to express the binding of oxygen to hemoglobin as a function of O2 concentration. Here [S] is the partial pressure of oxygen, which is directly proportional to dissolved [oxygen]. Y is fractional saturation (aka ). K is the concentration of oxygen that gives half-maximal saturation, and is equal to P50. h is the Hill coefficient and is an indication of the cooperativity of O2 binding.

Oxygen Binding to Myoglobin and Hemoglobin

Fig. 15. Hill plot for the binding of oxygen to myoglobin and hemoglobin. Plot is based on the Hill equation.

Effect of pH and CO2 Concentration on the Oxygen Affinity of Hemoglobin

Fig. 16. Effect of pH and CO2 concentration on the oxygen affinity of hemoglobin. Lowering the pH from 7.4 (red curve) to 7.2 (blue curve) results in the release of O2 from oxyhemoglobin. Raising the CO2 partial pressure from 0 to 40 (purple curve) also favors the release of O2 from oxyhemoglobin. In sum, protons and CO2 both shift the curve to the right (weaker binding of O2).

Transition from T to R State in Hemoglobin

Fig. 18. Ackers model for the cooperative transition in hemoglobin. Deoxygenated subunits have square corners, oxygenated have round corners. As oxygenation increases, strain within and between subunits increases. Whenever both dimers contain one or more oxy subunits, the T R transition occurs. Thus, tetramers in green are in T state, those in yellow are in R state.

SUMMARY
Binding of ligands to proteins involves hydrogen bonds, salt bridges, hydrophobic interactions, and van der Waals forces. The dissociation constant of a protein can be determined using fitting of the data to the equation defining the binding process or Scatchard analysis. Immunoglobulins bind antigens with very high affinity at binding sites that exhibit wide diversity. They have L2H2 tetrameric structure; the H chains have 4 domains, the L chains 2 domains. Each domain has antiparallel pleated sheet secondary structure.

Myoglobin and hemoglobin are the O2 transporters. Hemoglobin is an allosteric protein. They are mainly alpha-helical proteins.
The degree of cooperativity of ligand protein binding can be determined using a Hill plot. The cooperativity of hemoglobin has been described in the Ackers model.