4.1 Overview of Protein Structure in Chapter 4 The Three-Dimensional Structure of Proteins

4.1 Overview of Protein Structure

The possible conformations of a protein or protein segment include any structural state it can achieve without breaking covalent bonds. A change in conformation could occur, for example, by rotation about single bonds. However, of the many conformations that are theoretically possible in a protein containing hundreds of single bonds, one or a few generally predominate under biological conditions. The need for multiple stable conformations reflects the changes that must take place in most proteins as they bind to other molecules or catalyze reactions. The conformations existing under a given set of conditions are usually the ones that are thermodynamically the most stable — that is, having the lowest free energy (G). Proteins in any of their functional, folded conformations are often called native proteins.

For the vast majority of proteins, a particular structure or small set of structures is critical to function. However, in many cases, parts of proteins lack discernible structure. These protein segments are intrinsically disordered. In some cases, entire proteins are intrinsically disordered, yet are fully functional.

What determines the most stable conformations of a typical protein? We can build an understanding of protein conformation stepwise from the discussion of primary structure in Chapter 3 through a consideration of secondary, tertiary, and quaternary structures. To this approach we must add emphasis on common and classifiable folding patterns, variously called supersecondary structures, folds, or motifs, which provide an important organizational context to this complex endeavor.

A Protein’s Conformation Is Stabilized Largely by Weak Interactions

Stability is the tendency of a protein to maintain a native conformation. Native proteins are only marginally stable; the ΔG separating the folded and unfolded states in typical proteins under physiological conditions is in the range of only 5 to 65 kJ/mol. A given polypeptide chain can theoretically assume countless conformations, and as a result, the unfolded state of a protein is characterized by a high degree of conformational entropy. This entropy, along with the hydrogen-bonding interactions of many groups in the polypeptide chain with the solvent (water), tends to maintain the unfolded state. The chemical interactions that counteract these effects and stabilize the native conformation include disulfide (covalent) bonds and the weak (noncovalent) interactions and forces described in Chapter 2: hydrogen bonds, the hydrophobic effect, and ionic interactions.

Covalent disulfide bonds are strong, but they are also uncommon. The environment within most cells is highly reducing due to high concentrations of reductants such as glutathione, and most sulfhydryls will remain in the reduced state. Outside the cell, the environment is often more oxidizing, and disulfide formation is more likely to occur. In eukaryotes, disulfide bonds are found primarily in secreted, extracellular proteins (for example, the hormone insulin). Disulfide bonds are also uncommon in bacterial proteins. However, thermophilic bacteria, as well as the archaea, typically have many proteins with stabilizing disulfide bonds; this is presumably an adaptation to life at high temperatures.

For all proteins of all organisms, weak interactions are especially important in the folding of polypeptide chains into their secondary and tertiary structures. The association of multiple polypeptides to form quaternary structures also relies on these weak interactions.

About 200 to 460 kJ/mol are required to break a single covalent bond, whereas weak interactions can be disrupted by a mere 0.4 to 30 kJ/mol. Individual covalent bonds, such as disulfide bonds linking separate parts of a single polypeptide chain, are clearly much stronger than individual weak interactions. Yet, because they are so numerous, the weak interactions predominate as a stabilizing force in protein structure. In general, the protein conformation with the lowest free energy (that is, the most stable conformation) is the one with the maximum number of weak interactions.

The stability of a protein is not simply the sum of the free energies of formation of the many weak interactions within it. For every hydrogen bond formed in a protein during folding, a hydrogen bond (of similar strength) between the same group and water was broken. The net stability contributed by a given hydrogen bond, or the difference in free energies of the folded and unfolded states, may be close to zero. Ionic interactions may be either stabilizing or destabilizing. We must therefore look elsewhere to understand why a particular native conformation is favored.

Packing of Hydrophobic Amino Acids Away from Water Favors Protein Folding

On carefully examining the contribution of weak interactions to protein stability, we find that the hydrophobic effect generally predominates. Pure water contains a network of hydrogen-bonded $mathml alt text17$ $upper H Subscript 2 Baseline upper O$ molecules. No other molecule has the hydrogen-bonding potential of water, and the presence of other molecules in an aqueous solution disrupts the hydrogen bonding of water. When water surrounds a hydrophobic molecule, the optimal arrangement of hydrogen bonds results in a highly structured shell, or solvation layer, of water around the molecule (see Fig. 2-7). The increased order of the water molecules in the solvation layer correlates with an unfavorable decrease in the entropy of the water. However, when nonpolar groups cluster together, the extent of the solvation layer decreases, because each group no longer presents its entire surface to the solution. The result is a favorable increase in entropy. As described in Chapter 2, this increase in entropy is the major thermodynamic driving force for the association of hydrophobic groups in aqueous solution. Hydrophobic amino acid side chains therefore tend to cluster in a protein’s interior, away from water (think of an oil droplet in water). The amino acid sequences of most proteins thus include a significant content of hydrophobic amino acid side chains (especially Leu, Ile, Val, Phe, and Trp). These are positioned so that they are clustered when the protein is folded, forming a hydrophobic protein core.

Under physiological conditions, the formation of hydrogen bonds in a protein is driven largely by this same entropic effect. Polar groups can generally form hydrogen bonds with water and hence are soluble in water. However, the number of hydrogen bonds per unit mass is generally greater for pure water than for any other liquid or solution, and there are limits to the solubility of even the most polar molecules as their presence causes a net decrease in hydrogen bonding per unit mass. Therefore, a solvation layer forms to some extent even around polar molecules. Although the energy of formation of an intramolecular hydrogen bond between two polar groups in a macromolecule is largely canceled by the elimination of such interactions between these polar groups and water, the release of structured water as intramolecular associations form provides an entropic driving force for folding. Most of the net change in free energy as nonpolar amino acid side chains aggregate within a protein is therefore derived from the increased entropy in the surrounding aqueous solution resulting from the burial of hydrophobic surfaces. This more than counterbalances the large loss of conformational entropy as a polypeptide is constrained into its folded conformation.

Polar Groups Contribute Hydrogen Bonds and Ion Pairs to Protein Folding

The hydrophobic effect is clearly important in stabilizing conformation; the interior of a structured protein is generally a densely packed core of hydrophobic amino acid side chains. It is also important that any polar or charged groups in the protein interior have suitable partners for hydrogen bonding or ionic interactions. One hydrogen bond seems to contribute little to the stability of a native structure, but the presence of hydrogen-bonding groups without partners in the hydrophobic core of a protein can be so destabilizing that conformations containing these groups are often thermodynamically untenable. The favorable free-energy change resulting from the combination of several such groups with partners in the surrounding solution can be greater than the free-energy difference between the folded and unfolded states. In addition, hydrogen bonds between groups in a protein form cooperatively (formation of one makes formation of the next one more likely) in repeating secondary structures that optimize hydrogen bonding, as described below. In this way, hydrogen bonds often have an important role in guiding the protein-folding process.

The interaction of oppositely charged groups that form an ion pair, or salt bridge, can have either a stabilizing or destabilizing effect on protein structure. As in the case of hydrogen bonds, charged amino acid side chains interact with water and salts when the protein is unfolded, and the loss of those interactions must be considered when researchers evaluate the effect of a salt bridge on the overall stability of a folded protein. However, the strength of a salt bridge increases as it moves to an environment of lower dielectric constant, ε (p. 46): from the polar aqueous solvent (ε near 80) to the nonpolar protein interior (ε near 4). Salt bridges, especially those that are partly or entirely buried, can thus provide significant stabilization to a protein structure. This trend explains the increased occurrence of buried salt bridges in the proteins of thermophilic organisms. Ionic interactions also limit structural flexibility and confer a uniqueness to a particular protein structure that the clustering of nonpolar groups via the hydrophobic effect cannot provide.

Individual van der Waals Interactions Are Weak but Combine to Promote Folding

In the tightly packed atomic environment of a protein, one more type of weak interaction can have a significant effect: van der Waals interactions (p. 49). Van der Waals interactions are dipole-dipole interactions involving the permanent electric dipoles in groups such as carbonyls, transient dipoles derived from fluctuations of the electron cloud surrounding any atom, and dipoles induced by interaction of one atom with another that has a permanent or transient dipole. As atoms approach each other, these dipole-dipole interactions provide an attractive intermolecular force that operates over only a limited intermolecular distance (0.3 to 0.6 nm). Individually, van der Waals interactions contribute little to overall protein stability. However, in a well-packed protein, or in an interaction between a protein and another protein or other molecule at a complementary surface, the number of such interactions can be substantial.

Most of the structural patterns outlined in this chapter reflect two simple rules: (1) hydrophobic residues are largely buried in the protein interior, away from water, and (2) the number of hydrogen bonds and ionic interactions within the protein is maximized, thus reducing the number of unpaired hydrogen-bonding and ionic groups. Proteins within membranes (which we examine in Chapter 11) and proteins that are intrinsically disordered or have intrinsically disordered segments follow different rules. This reflects their particular function or environment, but weak interactions are still critical structural elements. For example, soluble but intrinsically disordered protein segments are often enriched in amino acid side chains that are charged (especially Arg, Lys, Glu) or small (Gly, Ala), providing little or no opportunity for the formation of a stable hydrophobic core.

The Peptide Bond Is Rigid and Planar

Covalent bonds, too, place important constraints on the conformation of a polypeptide. In the late 1930s, Linus Pauling and Robert Corey embarked on a series of studies that laid the foundation for our current understanding of protein structure. They began with a careful analysis of the peptide bond.

The α carbons of adjacent amino acid residues are separated by three covalent bonds, arranged as $mathml alt text21$ $upper C Subscript alpha Baseline em-dash upper C em-dash upper N em-dash upper C Subscript alpha Baseline period$ X-ray diffraction studies of crystals of amino acids and of simple dipeptides and tripeptides showed that the peptide $mathml alt text22$ $upper C em-dash upper N$ bond is somewhat shorter than the $mathml alt text23$ $upper C em-dash upper N$ bond in a simple amine and that the atoms associated with the peptide bond are coplanar. This indicated a resonance or partial sharing of two pairs of electrons between the carbonyl oxygen and the amide nitrogen (Fig. 4-2a). The oxygen has a partial negative charge and the hydrogen bonded to the nitrogen has a net partial positive charge, setting up a small electric dipole. The six atoms of the peptide group lie in a single plane, with the oxygen atom of the carbonyl group trans to the hydrogen atom of the amide nitrogen. From these findings Pauling and Corey concluded that the peptide $mathml alt text24$ $upper C em-dash upper N$ bonds, because of their partial double-bond character, cannot rotate freely. Rotation is permitted about the $mathml alt text25$ $upper N em-dash upper C Subscript alpha Baseline$ and the $mathml alt text26$ $upper C Subscript alpha Baseline em-dash upper C$ bonds. The backbone of a polypeptide chain can thus be pictured as a series of rigid planes, with consecutive planes sharing a common point of rotation at $mathml alt text27$ $upper C Subscript alpha$ (Fig. 4-2b). The rigid peptide bonds limit the range of conformations possible for a polypeptide chain.

A four-part figure, a, b, c, and d, shows the factors influencing the positioning of atoms forming a peptide bond. Part a shows resonance of the double bond, part b shows interactions between atoms in a polypeptide chain, part c shows interactions between C and N atoms in the chain, and part d shows a different view of interactions between C and N atoms in the chain. — FIGURE 4-2 The planar peptide group. (a) Each peptide bond has some double-bond character due to resonance and cannot rotate. Although the N atom in a peptide bond is often represented with a partial positive charge, careful consideration of bond orbitals and quantum mechanics indicates that the N has a net charge that is neutral or slightly negative. (b) Three bonds separate sequential α carbons in a polypeptide chain. The $mathml alt text29$ $upper N em-dash upper C Subscript alpha Baseline$ and $mathml alt text30$ $upper C Subscript alpha Baseline em-dash upper C$ bonds can rotate, described by dihedral angles designated ϕ and ψ, respectively. The peptide $mathml alt text33$ $upper C minus upper N$ bond is not free to rotate. Other single bonds in the backbone may also be rotationally hindered, depending on the size and charge of the R groups. (c) The atoms and planes defining ψ. (d) By convention, ϕ and ψ are $mathml alt text37$ $180 degree$ (or $mathml alt text38$ $negative 180 degree$ ) when the first and fourth atoms are farthest apart and the peptide is fully extended. As the viewer looks out along the bond undergoing rotation (from either direction), the ϕ and ψ angles increase as the fourth atom rotates clockwise relative to the first. In a protein, some of the conformations shown here (e.g., $mathml alt text41$ $0 degree$ ) are prohibited by steric overlap of atoms. In (b) through (d), the balls representing atoms are smaller than the van der Waals radii for this scale.

FIGURE 4-2 The planar peptide group. (a) Each peptide bond has some double-bond character due to resonance and cannot rotate. Although the N atom in a peptide bond is often represented with a partial positive charge, careful consideration of bond orbitals and quantum mechanics indicates that the N has a net charge that is neutral or slightly negative. (b) Three bonds separate sequential α carbons in a polypeptide chain. The $mathml alt text29$ $upper N em-dash upper C Subscript alpha Baseline$ and $mathml alt text30$ $upper C Subscript alpha Baseline em-dash upper C$ bonds can rotate, described by dihedral angles designated ϕ and ψ, respectively. The peptide $mathml alt text33$ $upper C minus upper N$ bond is not free to rotate. Other single bonds in the backbone may also be rotationally hindered, depending on the size and charge of the R groups. (c) The atoms and planes defining ψ. (d) By convention, ϕ and ψ are $mathml alt text37$ $180 degree$ (or $mathml alt text38$ $negative 180 degree$ ) when the first and fourth atoms are farthest apart and the peptide is fully extended. As the viewer looks out along the bond undergoing rotation (from either direction), the ϕ and ψ angles increase as the fourth atom rotates clockwise relative to the first. In a protein, some of the conformations shown here (e.g., $mathml alt text41$ $0 degree$ ) are prohibited by steric overlap of atoms. In (b) through (d), the balls representing atoms are smaller than the van der Waals radii for this scale.

Part a shows a piece of a polypeptide chain. There is a C alpha on the left that is further bonded on the left and bonded to C on the right that is double bonded to O above and further bonded to N on the right that is bonded to H below and further bonded to C alpha on the right that is further bonded. A double-headed arrow shows that this can interconvert with a similar molecule that has C alpha on the left that is further bonded on the left and bonded to C on the right that is single bonded to O minus above and double bonded to N plus on the right that is bonded to H below and further bonded to C alpha on the right that is further bonded. A double-headed arrow shows that this can interconvert with a similar molecule that has C alpha on the left that is further bonded on the left and bonded to C plus on the right that is single bonded to O minus bonded to N on the right that is bonded to H below and further bonded to C alpha on the right that is further bonded. Part b shows a ball-and-stick model of a series of amino acids with each placed in a rectangular plane. The amino terminus is on the left and the carboxyl terminus is on the right. The molecule begins with C alpha at the alpha terminus that is further bonded on the left and bonded to C on the right that is double bonded to O above and bonded to N on the right that is bonded to H below and further bonded to C alpha on the right. This structure repeats three more times to reach the carboxyl terminus. The bond between C alpha and C is 1.53 Angstroms, the double bond between C and O is 1.24 Angstroms, the bond between C and N is 1.32 Angstroms, and the bond between N and C alpha of the second amino acid is 1.46 Angstroms. There is a clockwise arrow around the bond between C alpha at the amino terminus and C to its right. There is also a clockwise arrow around the bond between the N to its right and C alpha of the second amino acid. This is labeled with Greek letter lowercase phi. Similar clockwise arrows are shown on each similar bond in the rest of the molecule. The one between the second C alpha and its adjacent C double bonded to O is labeled, Greek lowercase psi. There is a clockwise arrow around the bond between C alpha at the amino terminus and C to its right. Brackets extends from N to C alpha, from C alpha to C, and from the next C to N. C and N are shown to be in the rectangular plane. Part C shows N in a plane with an arrow pointing to C alpha above it in the same plane. An arrow with a clockwise circle around it labeled lowercase Greek letter psi points horizontally from C alpha to the next carbon, which is in another plane above and to the right. An arrow points from this carbon to the next N above. Part D shows N with an arrow pointing upward to C alpha. An arrow points from C alpha to another C atom behind it, from which an arrow points upward to N plus or minus 180 degrees. A clockwise circle of arrows runs from a faded atom behind C alpha label and labeled 0 degrees through faded circles at 60 degrees, 120 degrees, to the N at plus or minus 180 degrees at the 12 o’clock position, through minus 120 degrees, 60 degrees, and back to 0 degrees.

A photo of Linus Pauling. — Linus Pauling, 1901–1994

A photo of Robert Corey. — Robert Corey, 1897–1971

Peptide conformation is defined by three dihedral angles (also known as torsion angles) called ϕ (phi), ψ (psi), and ω (omega), reflecting rotation about each of the three repeating bonds in the peptide backbone. A dihedral angle is the angle at the intersection of two planes. In the case of peptides, the planes are defined by bond vectors in the peptide backbone. Two successive bond vectors describe a plane. Three successive bond vectors describe two planes (the central bond vector is common to both; Fig. 4-2c), and the angle between these two planes is what we measure to describe peptide conformation.

Key convention

The important dihedral angles in a peptide are defined by the three bond vectors connecting four consecutive main-chain (peptide backbone) atoms (Fig. 4-2c): ϕ involves the $mathml alt text46$ $upper C em-dash upper N em-dash upper C Subscript alpha Baseline em-dash upper C$ bonds (with the rotation occurring about the $mathml alt text47$ $upper N em-dash upper C Subscript alpha Baseline$ bond), and ψ involves the $mathml alt text49$ $upper N em-dash upper C Subscript alpha Baseline em-dash upper C em-dash upper N$ bonds. Both ϕ and ψ are defined as $mathml alt text52$ $plus-or-minus 180 degree$ when the polypeptide is fully extended and all peptide groups are in the same plane (Fig. 4-2d). As one looks down the central bond vector in the direction of the vector arrow (as depicted in Fig. 4-2c for ψ), the dihedral angles increase as the distal (fourth) atom is rotated clockwise (Fig. 4-2d). From the $mathml alt text54$ $plus-or-minus 180 degree$ position, the dihedral angle increases from $mathml alt text55$ $negative 180 degree$ to $mathml alt text56$ $0 degree comma$ at which point the first and fourth atoms are eclipsed. The rotation can be continued from $mathml alt text57$ $0 degree$ to $mathml alt text58$ $plus 180 degree$ (same position as $mathml alt text59$ $negative 180 degree$ ) to bring the structure back to the starting point. The third dihedral angle, ω, is not often considered. It involves the $mathml alt text61$ $upper C Subscript alpha Baseline em-dash upper C em-dash upper N em-dash upper C Subscript alpha Baseline$ bonds. The central bond in this case is the peptide bond, where rotation is constrained. The peptide bond is almost always (99.6% of the time) in the trans configuration, constraining ω to a value of $mathml alt text64$ $plus-or-minus 180 degree period$ For a rare cis peptide bond, $omega equals 0 degree period$ $omega equals 0 degree period$

In principle, ϕ and ψ can have any value between $mathml alt text68$ $negative 180 degree$ and $mathml alt text69$ $plus 180 degree comma$ but many values are prohibited by steric interference between atoms in the polypeptide backbone and amino acid side chains. The conformation in which both ϕ and ψ are $mathml alt text72$ $0 degree$ (Fig. 4-2d) is prohibited for this reason; this conformation is merely a reference point for describing the dihedral angles. Backbone angle preferences in a polypeptide represent yet another constraint on the overall folded structure of a protein.

SUMMARY 4.1 Overview of Protein Structure

A typical protein usually has one or more stable three-dimensional conformations that reflect its function. Some proteins have segments that are intrinsically disordered but are nonetheless essential for function.
Whereas nonpeptide covalent bonds, particularly disulfide bonds, can play a role in stabilization of some structures, proteins are stabilized largely by multiple weak, noncovalent interactions and forces.
The hydrophobic effect, derived from the increase in entropy of the surrounding water when nonpolar molecules or groups are clustered together, makes the major contribution to stabilizing the globular form of most soluble proteins.
Hydrogen bonds and ionic interactions are optimized in the thermodynamically most stable structures.
Van der Waals interactions involve attractive forces between molecular dipoles that occur over short distances. Individually these interactions are weak, but they combine in well-packed protein structures to provide significant effects and stabilization.
The nature of the covalent bonds in the polypeptide backbone places constraints on structure. The peptide bond has a partial double-bond character that keeps the entire six-atom peptide group in a rigid planar configuration. The $mathml alt text73$ $upper N em-dash upper C Subscript alpha Baseline$ and $mathml alt text74$ $upper C Subscript alpha Baseline em-dash upper C$ bonds can rotate to define the dihedral angles ϕ and ψ, respectively, although permitted values of ϕ and ψ are limited by steric clashes and other constraints.