Protein Interactions

Noncovalent molecular interactions occur between many different biomolecules and often involve proteins. For example, biological membranes are formed by the noncovalent associations of phospholipids and proteins. Also, glycosylated proteins and glycosylated lipids in cell membranes create a “sugar coat” on cell surfaces, where they assist cell aggregation {aggregare, to join together} and adhesion {adhaerere, to stick}.

Proteins play important roles in so many cell functions that we can consider them the “workhorses” of the body. Most soluble proteins fall into seven broad categories:

  1. Enzymes. Some proteins act as enzymes, biological catalysts that speed up chemical reactions. Enzymes play an important role in metabolism [discussed in Chapters 4 and 22].

  2. Membrane transporters. Proteins in cell membranes help move substances back and forth between the intracellular and extracellular compartments. These proteins may form channels in the cell membrane, or they may bind to molecules and carry them through the membrane. [Membrane transporters are discussed in detail in Chapter 5.].

  3. Signal molecules. Some proteins and smaller peptides act as hormones and other signal molecules. [Different types of signal molecules are described in Chapters 6 and 7.]

  4. Receptors. Proteins that bind signal molecules and initiate cellular responses are called receptors. [Receptors are discussed along with signal molecules in Chapter 6.]

  5. Binding proteins. These proteins, found mostly in the extracellular fluid, bind and transport molecules throughout the body. Examples you have already encountered include the oxygen-transporting protein hemoglobin and the cholesterol-binding proteins, such as LDL (low-density lipoprotein).

  6. Immunoglobulins. These extracellular immune proteins, also called antibodies, help protect the body from foreign invaders and substances. [Immune functions are discussed in Chapter 24.]

  7. Regulatory proteins. Regulatory proteins turn cell processes on and off or up and down. For example, the regulatory proteins known as transcription factors bind to DNA and alter gene expression and protein synthesis. The details of regulatory proteins can be found in cell biology textbooks.

Although soluble proteins are quite diverse, they do share some common features. They all bind to other molecules through noncovalent interactions. The binding, which takes place at a location on the protein molecule called a binding site, exhibits properties that will be discussed shortly: specificity, affinity, competition, and saturation. If binding of a molecule to the protein initiates a process, as occurs with enzymes, membrane transporters, and receptors, we can describe the activity rate of the process and the factors that modulate, or alter, the rate.

Any molecule or ion that binds to another molecule is called a ligand {ligare, to bind or tie}. Ligands that bind to enzymes and membrane transporters are also called substrates {sub-, below + stratum, a layer}. Protein signal molecules and protein transcription factors are ligands. Immunoglobulins bind ligands, but the immunoglobulin-ligand complex itself then becomes a ligand [for details, see Chapter 24].

Proteins Are Selective about the Molecules They Bind

The ability of a protein to bind to a certain ligand or a group of related ligands is called specificity. Some proteins are very specific about the ligands they bind, while others bind to whole groups of molecules. For example, the enzymes known as peptidases bind polypeptide ligands and break apart peptide bonds, no matter which two amino acids are joined by those bonds. For this reason peptidases are not considered to be very specific in their action. In contrast, aminopeptidases also break peptide bonds but are more specific. They will bind only to one end of a protein chain (the end with an unbound amino group) and can act only on the terminal peptide bond.

Ligand binding requires molecular complementarity. In other words, the ligand and the protein binding site must be complementary, or compatible. In protein binding, when the ligand and protein come close to each other, noncovalent interactions between the ligand and the protein’s binding site allow the two molecules to bind. From studies of enzymes and other binding proteins, scientists have discovered that a protein’s binding site and the shape of its ligand do not need to fit one another exactly. When the binding site and the ligand come close to each other, they begin to interact through hydrogen and ionic bonds and van der Waals forces. The protein’s binding site then changes shape (conformation) to fit more closely to the ligand. This induced-fit model of protein-ligand interaction is shown in Figure 2.10.

FIG. 2.10 The induced-fit model of protein-ligand (L) binding

Protein-Binding Reactions Are Reversible

The degree to which a protein is attracted to a ligand is called the protein’s affinity for the ligand. If a protein has a high affinity for a given ligand, the protein is more likely to bind to that ligand than to a ligand for which the protein has a lower affinity.

Protein binding to a ligand can be written using the same notation that we use to represent chemical reactions:

P + L PL

where P is the protein, L is the ligand, and PL is the bound protein-ligand complex. The double arrow indicates that binding is reversible.

Reversible binding reactions go to a state of equilibrium, where the rate of binding ( P + L PL ) is exactly equal to the rate of unbinding, or dissociation ( P + L PL ) When a reaction is at equilibrium, the ratio of the product concentration, or protein-ligand complex [PL], to the reactant concentrations [P][L] is always the same. This ratio is called the equilibrium constant Keq, and it applies to all reversible chemical reactions:

K eq = [ PL ] [ P][L ]

The square brackets [ ] around the letters indicate concentrations of the protein, ligand, and protein-ligand complex.

Binding Reactions Obey the Law of Mass Action

Equilibrium is a dynamic state. In the living body, concentrations of protein or ligand change constantly through synthesis, breakdown, or movement from one compartment to another. What happens to equilibrium when the concentration of P or L changes? The answer to this question is shown in Figure 2.11, which begins with a reaction at equilibrium (Fig. 2.11a).

FIG. 2.11 The law of mass action

In Figure 2.11b, the equilibrium is disturbed when more protein or ligand is added to the system. Now the ratio of [PL] to [P][L] differs from the Keq. In response, the rate of the binding reaction increases to convert some of the added P or L into the bound protein-ligand complex (Fig. 2.11c). As the ratio approaches its equilibrium value again, the rate of the forward reaction slows down until finally the system reaches the equilibrium ratio once more (Fig. 2.11d). [P], [L], and [PL] have all increased over their initial values, but the equilibrium ratio has been restored.

The situation just described is an example of a reversible reaction obeying the law of mass action, a simple relationship that holds for chemical reactions whether in a test tube or in a cell. You may have learned this law in chemistry as Le Châtelier’s principle. In very general terms, the law of mass action says that when a reaction is at equilibrium, the ratio of the products to the substrates is always the same. If the ratio is disturbed by adding or removing one of the participants, the reaction equation will shift direction to restore the equilibrium condition. (Note that the law of mass action is not the same as mass balance [see Chapter 1 here].)

One example of this principle at work is the transport of steroid hormones in the blood. Steroids are hydrophobic, so more than 99% of hormone in the blood is bound to carrier proteins. The equilibrium ratio [PL]/[P][L] is 99% bound/1% unbound hormone. However, only the unbound or “free” hormone can cross the cell membrane and enter cells. As unbound hormone leaves the blood, the equilibrium ratio is disturbed. The binding proteins then release some of the bound hormone until the 99/1 ratio is again restored. The same principle applies to enzymes and metabolic reactions. Changing the concentration of one participant in a chemical reaction has a chain-reaction effect that alters the concentrations of other participants in the reaction.

Concept Check

  1. Consider the carbonic acid reaction, which is reversible:

    CO 2 + H 2 O H 2 CO 3 (carbonic acid) H + + HCO 3

    If the carbon dioxide concentration in the body increases, what happens to the concentration of carbonic acid (H2CO3)? What happens to the pH?

The Dissociation Constant Indicates Affinity

In protein-binding reactions, the equilibrium constant is a quantitative representation of the protein’s binding affinity for the ligand: high affinity for the ligand means a larger Keq. The reciprocal of the equilibrium constant is called the dissociation constant (Kd).

K d = [ P ] [ L ] [ PL ]

A large K d indicates low binding affinity of the protein for the ligand and more P and L remaining in the unbound state. Conversely, a small K d means a higher value for [PL] relative to [P] and [L], so a small K d indicates higher affinity of the protein for the ligand.

If one protein binds to several related ligands, a comparison of their Kd values can tell us which ligand is more likely to bind to the protein. The related ligands compete for the binding sites and are said to be competitors. Competition between ligands is a universal property of protein binding.

Competing ligands that mimic each other’s actions are called agonists {agonist, contestant}. Agonists may occur in nature, such as nicotine, the chemical found in tobacco, which mimics the activity of the neurotransmitter acetylcholine by binding to the same receptor protein. Agonists can also be synthesized using what scientists learn from the study of protein–ligand binding sites. The ability of agonist molecules to mimic the activity of naturally occurring ligands has led to the development of many drugs.

Concept Check

  1. A researcher is trying to design a drug to bind to a particular cell receptor protein. Candidate molecule A has a Kd of 4.9 for the receptor. Molecule B has a Kd of 0.3. Which molecule has the most potential to be successful as the drug?

Multiple Factors Alter Protein Binding

A protein’s affinity for a ligand is not always constant. Chemical and physical factors can alter, or modulate, binding affinity or can even totally eliminate it. Some proteins must be activated before they have a functional binding site. In this section we discuss some of the processes that have evolved to allow activation, modulation, and inactivation of protein binding.

Isoforms

Closely related proteins whose function is similar but whose affinity for ligands differs are called isoforms of one another. For example, the oxygen-transporting protein hemoglobin has multiple isoforms. One hemoglobin molecule has a quaternary structure consisting of four subunits (see Fig. 2.3). In the developing fetus, the hemoglobin isoform has two α (alpha) chains and two γ (gamma) chains that make up the four subunits. Shortly after birth, fetal hemoglobin molecules are broken down and replaced by adult hemoglobin. The adult hemoglobin isoform retains the two a chain isoforms but has two β (beta) chains in place of the γ chains. Both adult and fetal isoforms of hemoglobin bind oxygen, but the fetal isoform has a higher affinity for oxygen. This makes it more efficient at picking up oxygen across the placenta.

Activation

Some proteins are inactive when they are synthesized in the cell. Before such a protein can become active, enzymes must chop off one or more portions of the molecule (Fig. 2.12a). Protein hormones (a type of signal molecule) and enzymes are two groups that commonly undergo such proteolytic activation {lysis, to release}. The inactive forms of these proteins are often identified with the prefix pro- {before}: prohormone, proenzyme, proinsulin, for example. Some inactive enzymes have the suffix -ogen added to the name of the active enzyme instead, as in trypsinogen, the inactive form of trypsin.

The activation of some proteins requires the presence of a cofactor, which is an ion or small organic functional group. Cofactors must attach to the protein before the binding site will become active and bind to ligand (Fig. 2.12b). Ionic cofactors include Ca2+, Mg2+, and Fe2+. Many enzymes will not function without their cofactors.

Modulation

The ability of a protein to bind a ligand and initiate a response can be altered by various factors, including temperature, pH, and molecules that interact with the protein. A factor that influences either protein binding or protein activity is called a modulator. There are two basic mechanisms by which modulation takes place. The modulator either (1) changes the protein’s ability to bind the ligand or it (2) changes the protein’s activity or its ability to create a response. Table 2.3 summarizes the different types of modulation.

Table 2.3 Factors that Affect Protein Binding

Essential for Binding Activity
Cofactors Required for ligand binding at binding site
Proteolytic activation Converts inactive to active form by removing part of molecule. Examples: digestive enzymes, protein hormones
Modulators and Factors that alter Binding or Activity
Competitive inhibitor Competes directly with ligand by binding reversibly to active site
Irreversible inhibitor Binds to binding site and cannot be displaced
Allosteric modulator Binds to protein away from binding site and changes activity; may be inhibitors or activators
Covalent modulator Binds covalently to protein and changes its activity. Example: phosphate groups
pH and temperature Alter three-dimensional shape of protein by disrupting hydrogen or S–S bonds; may be irreversible if protein becomes denatured

Chemical modulators are molecules that bind covalently or noncovalently to proteins and alter their binding ability or their activity. Chemical modulators may activate or enhance ligand binding, decrease binding ability, or completely inactivate the protein so that it is unable to bind any ligand. Inactivation may be either reversible or irreversible.

Antagonists, also called inhibitors, are chemical modulators that bind to a protein and decrease its activity. Many are simply molecules that bind to the protein and block the binding site without causing a response. They are like the guy who slips into the front of the movie ticket line to chat with his girlfriend, the cashier. He has no interest in buying a ticket, but he prevents the people in line behind him from getting their tickets for the movie.

Competitive inhibitors are reversible antagonists that compete with the customary ligand for the binding site (Fig. 2.12d). The degree of inhibition depends on the relative concentrations of the competitive inhibitor and the customary ligand, as well as on the protein’s affinities for the two. The binding of competitive inhibitors is reversible: increasing the concentration of the customary ligand can displace the competitive inhibitor and decrease the inhibition.

Irreversible antagonists, on the other hand, bind tightly to the protein and cannot be displaced by competition. Antagonist drugs have proven useful for treating many conditions. For example, tamoxifen, an antagonist to the estrogen receptor, is used in the treatment of hormone-dependent cancers of the breast.

Allosteric and covalent modulators may be either antagonists or activators. Allosteric modulators {allos, other + stereos, solid (as a shape)} bind reversibly to a protein at a regulatory site away from the binding site, and by doing so change the shape of the binding site. Allosteric inhibitors are antagonists that decrease the affinity of the binding site for the ligand and inhibit protein activity (Fig. 2.12e). Allosteric activators increase the probability of protein-ligand binding and enhance protein activity (Fig. 2.12c). For example, the oxygen-binding ability of hemoglobin changes with allosteric modulation by carbon dioxide, H+, and several other factors [see Chapter 18].

Covalent modulators are atoms or functional groups that bind covalently to proteins and alter the proteins’ properties. Like allosteric modulators, covalent modulators may either increase or decrease a protein’s binding ability or its activity. One of the most common covalent modulators is the phosphate group. Many proteins in the cell can be activated or inactivated when a phosphate group forms a covalent bond with them, the process known as phosphorylation.

One of the best known chemical modulators is the antibiotic penicillin. Alexander Fleming discovered this compound in 1928, when he noticed that Penicillium mold inhibited bacterial growth in a petri dish. By 1938, researchers had extracted the active ingredient penicillin from the mold and used it to treat infections in humans. Yet it was not until 1965 that researchers figured out exactly how the antibiotic works. Penicillin is an antagonist that binds to a key bacterial protein by mimicking the normal ligand. Because penicillin forms unbreakable bonds with the protein, the protein is irreversibly inhibited. Without the protein, the bacterium is unable to make a rigid cell wall. With no rigid cell wall, the bacterium swells, ruptures, and dies.

Physical Factors

Physical conditions such as temperature and pH (acidity) can have dramatic effects on protein structure and function. Small changes in pH or temperature act as modulators to increase or decrease activity (Fig. 2.13a). However, once these factors exceed some critical value, they disrupt the noncovalent bonds holding the protein in its tertiary conformation. The protein loses its shape and, along with that, its activity. When the protein loses its conformation, it is said to be denatured.

If you have ever fried an egg, you have watched this transformation happen to the egg white protein albumin as it changes from a slithery clear state to a firm white state. Hydrogen ions in high enough concentration to be called acids have a similar effect on protein structure. During preparation of ceviche, the national dish of Ecuador, raw fish is marinated in lime juice. The acidic lime juice contains hydrogen ions that disrupt hydrogen bonds in the muscle proteins of the fish, causing the proteins to become denatured. As a result, the meat becomes firmer and opaque, just as it would if it were cooked with heat.

In a few cases, activity can be restored if the original temperature or pH returns. The protein then resumes its original shape as if nothing had happened. Usually, however, denaturation produces a permanent loss of activity. There is certainly no way to unfry an egg or uncook a piece of fish. The potentially disastrous influence of temperature and pH on proteins is one reason these variables are so closely regulated by the body.

Concept Check

  1. Match each chemical to its action(s).

    • (a) Allosteric modulator

    • (b) Competitive inhibitor

    • (c) Covalent modulator

    1. Bind away from the binding site

    2. Bind to the binding site

    3. Inhibit activity only

    4. Inhibit or enhance activity

The Body Regulates the Amount of Protein in Cells

The final characteristic of proteins in the human body is that the amount of a given protein varies over time, often in a regulated fashion. The body has mechanisms that enable it to monitor whether it needs more or less of certain proteins. Complex signaling pathways, many of which themselves involve proteins, direct particular cells to make new proteins or to break down (degrade) existing proteins. This programmed production of new proteins (receptors, enzymes, and membrane transporters, in particular) is called up-regulation. Conversely, the programmed removal of proteins is called down-regulation. In both instances, the cell is directed to make or remove proteins to alter its response.

The amount of protein present in a cell has a direct influence on the magnitude of the cell’s response. For example, the graph in Figure 2.13b shows the results of an experiment in which the amount of ligand is held constant while the amount of protein is varied. As the graph shows, an increase in the amount of protein present causes an increase in the response.

As an analogy, think of the checkout lines in a supermarket. Imagine that each cashier is an enzyme, the waiting customers are ligand molecules, and people leaving the store with their purchases are products. One hundred customers can be checked out faster when there are 25 lines open than when there are only 10 lines. Likewise, in an enzymatic reaction, the presence of more protein molecules (enzyme) means that more binding sites are available to interact with the ligand molecules. As a result, the ligands are converted to products more rapidly.

Regulating protein concentration is an important strategy that cells use to control their physiological processes. Cells alter the amount of a protein by influencing both its synthesis and its breakdown. If protein synthesis exceeds breakdown, protein accumulates and the reaction rate increases. If protein breakdown exceeds synthesis, the amount of protein decreases, as does the reaction rate. Even when the amount of protein is constant, there is still a steady turnover of protein molecules.

Reaction Rate Can Reach a Maximum

If the concentration of a protein in a cell is constant, then the concentration of the ligand determines the magnitude of the response. Fewer ligands activate fewer proteins, and the response is low. As ligand concentrations increase, so does the magnitude of the response, up to a maximum where all protein binding sites are occupied.

Figure 2.13c shows the results of a typical experiment in which the protein concentration is constant but the concentration of ligand varies. At low ligand concentrations, the response rate is directly proportional to the ligand concentration. Once the concentration of ligand molecules exceeds a certain level, the protein molecules have no more free binding sites. The proteins are fully occupied, and the rate reaches a maximum value. This condition is known as saturation. Saturation applies to enzymes, membrane transporters, receptors, binding proteins, and immunoglobulins.

An analogy to saturation appeared in the early days of television on the I Love Lucy show. Lucille Ball was working at the conveyor belt of a candy factory, loading chocolates into the little paper cups of a candy box. Initially, the belt moved slowly, and she had no difficulty picking up the candy and putting it into the box. Gradually, the belt brought candy to her more rapidly, and she had to increase her packing speed to keep up. Finally, the belt brought candy to her so fast that she could not pack it all in the boxes because she was working at her maximum rate. That was Lucy’s saturation point. (Her solution was to stuff the candy into her mouth as well as into the box!)

In conclusion, you have now learned about the important and nearly universal properties of soluble proteins: shape-function relationships, ligand binding, saturation, specificity, competition, and activation/inhibition. You will revisit these concepts many times as you work through the organ systems of the body. The body’s insoluble proteins, which are key structural components of cells and tissues, are covered in the next chapter.

Concept Check

  1. What happens to the rate of an enzymatic reaction as the amount of enzyme present decreases?

  2. What happens to the rate of an enzymatic reaction when the enzyme has reached saturation?