CHEM 7784 Biochemistry Professor Bensley Chapter 5.1: Protein Function - Reversible Binding of Protein to a Ligand CHEM 7784 Biochemistry Professor Bensley

CHAPTER 5.1 Reversible Binding of Protein to a Ligand Today’s Objectives - To learn and understand: Reversible binding of ligands Structure of myoglobin and hemoglobin Origin of cooperativity in hemoglobin

Functions of Globular Proteins Storage of ions and molecules myoglobin, ferritin Transport of ions and molecules hemoglobin, serotonin transporter Defense against pathogens antibodies, cytokines Muscle contraction actin, myosin Biological catalysis chymotrypsin, lysozyme

FIGURE 4-15a,b Tertiary structure of sperm whale myoglobin FIGURE 4-15a,b Tertiary structure of sperm whale myoglobin. (PDB ID 1MBO) Orientation of the protein is similar in (a) through (d); the heme group is shown in red. In addition to illustrating the myoglobin structure, this figure provides examples of several different ways to display protein structure. (a) The polypeptide backbone in a ribbon representation of a type introduced by Jane Richardson, which highlights regions of secondary structure. The α-helical regions are evident. (b) Surface contour image; this is useful for visualizing pockets in the protein where other molecules might bind.

Binding: Quantitative Description Consider a process in which a ligand (L) binds reversibly to a site in the protein (P) The equilibrium composition is characterized by the equilibrium constant Ka ka + L PL P kd

Binding: Analysis in Terms of the Bound Fraction In practice, we can often determine the fraction of occupied binding sites Substituting [PL] with Ka[L][P], we’ll eliminate [PL] Eliminating [P] and rearranging gives the result in terms of equilibrium association constant: In terms of the more commonly used equilibrium dissociation constant:

Binding: Graphical Analysis The fraction of bound sites depends on the free ligand concentration and Kd In a typical experiment, ligand concentration is the known independent variable Kd can be determined graphically or via least-squares regression [L]  [L]total FIGURE 5-4a Graphical representations of ligand binding. The fraction of ligand-binding sites occupied, θ, is plotted against the concentration of free ligand. Both curves are rectangular hyperbolas. (a) A hypothetical binding curve for a ligand L. The [L] at which half of the available ligand-binding sites are occupied is equivalent to 1/Ka, or Kd. The curve has a horizontal asymptote at θ = 1 and a vertical asymptote (not shown) at [L] = –1/Ka.

FIGURE 5-4 Graphical representations of ligand binding FIGURE 5-4 Graphical representations of ligand binding. The fraction of ligand-binding sites occupied, θ, is plotted against the concentration of free ligand. Both curves are rectangular hyperbolas. (b) A curve describing the binding of oxygen to myoglobin. The partial pressure of O2 in the air above the solution is expressed in kilopascals (kPa). Oxygen binds tightly to myoglobin, with a P50 of only 0.26 kPa.

Specificity: Lock-and-Key Model “Lock and Key” model by Emil Fischer (1894) assumes that complementary surfaces are preformed. + FIGURE 6-4 Complementary shapes of a substrate and its binding site on an enzyme. The enzyme dihydrofolate reductase with its substrate NADP+ (red), unbound (top) and bound (bottom); another bound substrate, tetrahydrofolate (yellow), is also visible (PDB ID 1RA2). In this model, the NADP+ binds to a pocket that is complementary to it in shape and ionic properties, an illustration of Emil Fischer's "lock and key" hypothesis of enzyme action. In reality, the complementarity between protein and ligand (in this case substrate) is rarely perfect, as we saw in Chapter 5.

Specificity: Induced Fit Conformational changes may occur upon ligand binding (Daniel Koshland in 1958). This adaptation is called the induced fit. Induced fit allows for tighter binding of the ligand Induced fit can increase the affinity of the protein for a second ligand Both the ligand and the protein can change their conformations +

Myoglobin/ Hemoglobin First protein structures determined Oxygen carriers Hemoglobin: transports O2 from lungs to tissues Myoglobin: O2 storage protein

Mb and Hb Subunits Structurally Similar 8 alpha-helices Contain heme group Mb monomeric protein Hb heterotetramer (α2β2) myoglobin hemoglobin

Heme Group FIGURE 5-1d Heme. The iron atom of heme has six coordination bonds: four in the plane of, and bonded to, the flat porphyrin ring system, and (d) two perpendicular to it.

Structure of Myoglobin FIGURE 5-2 The heme group viewed from the side. This view shows the two coordination bonds to Fe2+ that are perpendicular to the porphyrin ring system. One is occupied by a His residue, sometimes called the proximal His; the other is the binding site for oxygen. The remaining four coordination bonds are in the plane of, and bonded to, the flat porphyrin ring system.

FIGURE 5-3 Myoglobin. (PDB ID 1MBO) The eight α-helical segments (shown here as cylinders) are labeled A through H. Nonhelical residues in the bends that connect them are labeled AB, CD, EF, and so forth, indicating the segments they interconnect. A few bends, including BC and DE, are abrupt and do not contain any residues; these are not normally labeled. (The short segment visible between D and E is an artifact of the computer representation.) The heme is bound in a pocket made up largely of the E and F helices, although amino acid residues from other segments of the protein also participate.

Hemoglobin FIGURE 5-6 Comparison of the structures of myoglobin (PDB ID 1MBO) and the β subunit of hemoglobin (derived from PDB ID 1HGA).

Oxygen Binding Curves Mb has hyberbolic O2 binding curve Mb binds O2 tightly. Releases at very low pO2 Hb has sigmoidal O2 binding curve Hb high affinity for O2 at high pO2 (lungs) Hb low affinity for O2 at low pO2 (tissues)

Oxygen Binding Curve

Oxygen Binding Curve

O2 Binding to Hb shows Positive Cooperativity Hb binds four O2 molecules O2 affinity increases as each O2 molecule binds Increased affinity due to conformation change Deoxygenated form = T (tense) form = low affinity Oxygenated form = R (relaxed) form = high affinity

O2 Binding to Hb shows Positive Cooperativity

Conformational Change is Triggered by Oxygen Binding FIGURE 5-11 Changes in conformation near heme on O2 binding to deoxyhemoglobin. (Derived from PDB ID 1HGA and 1BBB) The shift in the position of helix F when heme binds O2 is thought to be one of the adjustments that triggers the T → R transition.

FIGURE 5-10 The T R transition FIGURE 5-10 The T R transition. (PDB ID 1HGA and 1BBB) In these depictions of deoxyhemoglobin, as in Figure 5-9, the α subunits are blue and the β subunits are gray. Positively charged side chains and chain termini involved in ion pairs are shown in blue, their negatively charged partners in red. The Lys C5 of each α subunit and Asp FG1 of each β subunit are visible but not labeled (compare Figure 5-9a). Note that the molecule is oriented slightly differently than in Figure 5-9. The transition from the T state to the R state shifts the subunit pairs substantially, affecting certain ion pairs. Most noticeably, the His HC3 residues at the carboxyl termini of the β subunits, which are involved in ion pairs in the T state, rotate in the R state toward the center of the molecule, where they are no longer in ion pairs. Another dramatic result of the T → R transition is a narrowing of the pocket between the β subunits. Video on Hemoglobin

FIGURE 5-16 Effect of pH on oxygen binding to hemoglobin FIGURE 5-16 Effect of pH on oxygen binding to hemoglobin. The pH of blood is 7.6 in the lungs and 7.2 in the tissues. Experimental measurements on hemoglobin binding are often performed at pH 7.4.

Allosteric Interactions Allosteric interaction occurs when specific molecules bind a protein and modulate activity Allosteric modulators or allosteric effectors Bind reversibly to site separate from functional binding or active site Modulation of activity occurs through change in protein conformation 2,3 bisphosphoglycerate (BPG), CO2 and protons are allosteric effectors of Hb binding of O2

Regulation of O2 Binding by 2,3-Bisphospho-glycerate FIGURE 5-17 Effect of BPG on oxygen binding to hemoglobin. The BPG concentration in normal human blood is about 5 mM at sea level and about 8 mM at high altitudes. Note that hemoglobin binds to oxygen quite tightly when BPG is entirely absent, and the binding curve seems to be hyperbolic. In reality, the measured Hill coefficient for O2-binding cooperativity decreases only slightly (from 3 to about 2.5) when BPG is removed from hemoglobin, but the rising part of the sigmoid curve is confined to a very small region close to the origin. At sea level, hemoglobin is nearly saturated with O2 in the lungs, but just over 60% saturated in the tissues, so the amount of O2 released in the tissues is about 38% of the maximum that can be carried in the blood. At high altitudes, O2 delivery declines by about one-fourth, to 30% of maximum. An increase in BPG concentration, however, decreases the affinity of hemoglobin for O2, so approximately 37% of what can be carried is again delivered to the tissues.