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Wednesday, January 31, 2018

Chapter 6 | The Three-Dimensional Structure of Proteins Introduction - First, the chain appears to be locally coiled into regions of helical structure. • Such local regular folding is called the secondary structure æ of the molecule • We call this further level of three-dimensional organization the tertiary structure æ of the molecule • The arrangement of subunits defines the quaternary structure æ of a multisubunit protein • Concept: Protein molecules have four levels of structural organization: primary (sequence), secondary (local folding), tertiary (overall folding), and quaternary (subunit association).

6.1 | Secondary Structure: Regular Ways to Fold the Polypeptide Chain - Theoretical Descriptions of Regular Polypeptide Structures • 1. The bond lengths and bond angles should be distorted as little as possible from those found through X-ray diffraction studies of amino acids and peptides, as shown in Figure 5.10(b) æ.

• 2. No two atoms should approach one another more closely than is allowed by their van der Waals radii; thus, there are steric restrictions to rotations around the bonds that make up the peptide backbone (see Figure 6.3 æ).

• 3. The six atoms in the peptide amide group must remain coplanar with the associated α-carbons in the trans configuration, as shown in Figure 5.10(b) æ. Consequently, rotation is possible only about the two bonds adjacent to the α-carbon in each amino acid residue, as shown in Figure" 6.3 æ.

• 4. Some kind of noncovalent bonding is necessary to stabilize a regular folding. The most obvious possibility is hydrogen bonding between amide protons and carbonyl oxygens:

- α Helices and β Sheets • The two structures they proposed as most likely—the right-handed α helix, and the β sheet–are shown in Figure 6.4(a) æ and (b) æ.

• These two structures are, in fact, the most commonly observed secondary structures in proteins. Figure 6.4(c) æ shows the so-called 310 helix, which is observed in some proteins but is not as common as the α helix

• Concept: Of the several possible secondary structures for polypeptides, the most frequently observed are the α helix and the β sheet

- Describing the Structures: Helices and Sheets • A β sheet is composed of two or more β strands with main-chain hydrogen bonds between adjacent β strands • The β sheet shown in Figure 6.4(b) æ shows two β strands arranged such that the N-terminus to C-terminus orientations of the two strands are in opposite directions. Such an arrangement of strands is called antiparallel, whereas the arrangement with both strands oriented in the same direction is called parallel

• There is one more regularly repeating conformation that is commonly observed in protein structures—the socalled polyproline II helix

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Wednesday, January 31, 2018 - Amphipathic Helices and Sheets • In an α helix, the side chain of each amino acid residue points outward, away from the center of the helix (Figure 6.7 æ).

- In a β sheet, a network of main-chain hydrogen bonds connects the β strands - Secondary structures that display a predominantly hydrophobic face opposite a predominantly hydrophilic face are said to be “amphipathic æ” (or “amphiphilic”).

• Concept: An amphiphilic α helix will have side chains of similar polarity every 3–4 residues, whereas a β strand in an amphiphilic β sheet will have alternating polar and nonpolar side chains.

• The combinations of φ and ψ angles that are most favorable (or “allowed”)— because they relieve steric crowding—are shown in a systematic description of polypeptide backbone conformation called a Ramachandran plot æ

- Ramachandran Plots • Each residue in a polypeptide chain has two backbone bonds about which rotation is permitted. The angles of rotation about these bonds, defined as φ (phi) and ψ (psi), describe the backbone conformation of any particular residue in any protein

• The backbone conformation of any particular residue in a protein can be described by a point on a map (Figure 6.9 æ) with coordinates φ and ψ. Such maps are called Ramachandran plots

• Concept: The Ramachandran plot illustrates which combinations of φ and ψ angles are sterically allowed in regular repeats of secondary structure.

• Although most of the points fall in “allowed” regions, a few lie in “nonallowed” regions. These are mainly glycines, for which a much wider range of φ and ψ angles is allowed because the side chain is so small

• We begin with the observation that two major classes of proteins exist. These are called fibrous and globular proteins and are distinguished by major structural differences

6.2 | Fibrous Proteins: Structural Materials of Cells and Tissues - Fibrous proteins æ are distinguished from globular proteins by their filamentous, or elongated, form. - Concept: Fibrous proteins are elongated molecules with well-defined secondary structures. They usually play structural roles in the cell.

- The Keratins • Two important classes of fibrous proteins that have similar amino acid sequences and biological function are called α- and β-keratins.

• The α-keratins æ are the major proteins of hair and fingernails, and comprise a major fraction of animal skin. • The α-keratins are members of a broad group of intermediate filament proteins, which play important structural roles in the nuclei, cytoplasm, and surfaces of many cell types

• Individual α-keratin molecules contain long sequences–over 300 residues–that are wholly α-helical. Pairs of these right-handed helices twist about one another in the left-handed coiled-coil structure

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Wednesday, January 31, 2018 • The α-keratin molecule includes a small globular region that is covalently attached to the coiled-coil sequence. In keratin intermediate filaments, pairs of coiled coils tend to associate into stretchy and flexible fibrils

- Concept: α-Keratin is built on a coiled-coil α-helical structure. • The β-keratins, as their name implies, contain much more β-sheet structure - Fibroin • The β-sheet structure is most elegantly utilized in the fibers spun by silkworms and spiders • Concept: Fibroin is a β sheet protein. Almost half of its residues are glycine - Collagen • Because it performs such a wide variety of functions, collagen is the most abundant single protein in most vertebrates

• The basic unit of the collagen fiber is the tropocollagen molecule, a triple helix of three polypeptide chains, each about 1000 residues in length

• The conformation of the left-handed collagen helix is favored by the presence of proline or hydroxyproline in the tropocollagen molecule

• Concept: Collagen fibers are built from triple helices of polypeptides rich in glycine and proline. • Collagen is also unusual in its widespread modification of proline to hydroxyproline. - Most of the hydrogen bonds between chains in the triple helix are from amide protons to carbonyl oxygens, but the —OH groups of hydroxyproline also seem to participate in stabilizing the fiber.

• The enzymes that catalyze the hydroxylations of proline and lysine residues in collagen require vitamin C, Lascorbic acid. A symptom of extreme vitamin C deficiency, called scurvy, is the weakening of collagen fibers caused by the failure to hydroxylate these side chains, which results in reduced hydrogen bonding between chains in the collagen fiber

- Connection: Scurvy is caused by vitamin C deficiency, which leads to failure to hydroxylate prolines and lysines in collagen.

• The individual tropocollagen molecules pack together in a collagen fiber in a specific way • Part of the toughness of collagen is due to the cross-linking of tropocollagen molecules to one another via a reaction involving lysine side chains

- Connection: Collagen becomes more crosslinked, and therefore more brittle, with age. • First, proteins have evolved to serve a diversity of functions. - Second, the fibrous proteins do this by taking advantage of the propensities of particular repetitive sequences of amino acid residues to favor one kind of secondary structure or another.

- Finally, post-translational modification of proteins, including cross-linking, is an important adjunct in tailoring a protein to its function

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Wednesday, January 31, 2018 6.3 | Globular Proteins: Tertiary Structure and Functional Diversity - Different Folding for Different Functions • Most of the chemical work of the cell–the synthesizing, transporting, and metabolizing–is mediated by the enormously varied globular proteins æ.

- These proteins are so named because their polypeptide chains are folded into compact structures very unlike the extended, filamentous forms of the fibrous proteins

• Within the protein molecule, the polypeptide chain is often locally folded into one or another of the kinds of secondary structure (α helix, β sheet, and so forth) that we have already discussed.

- But to make the structure globular and compact, these regions of secondary structure must themselves be folded on one another.

- This packing together of the protein chain defines the tertiary structure æ of the protein • Structure defines function - We have learned a great deal about the biochemistry that occurs in cells as a result of the detailed structural analysis of protein molecules, largely through the use of X-ray diffraction methods (see Tools of Biochemistry 4B æ) and nuclear magnetic resonance spectroscopy æ or NMR

- Concept: Globular proteins not only possess secondary structures but also are folded into compact tertiary structures.

- Different Modes of Display Aid Our Understanding of Protein Structure • In spite of being a very small protein, ubiquitin has a complex molecular structure due to the 1231 atoms distributed among its 76 amino acids

• In most cases, it is the outer surface of the protein that is of greatest importance in defining its function. - Varieties of Globular Protein Structure: Patterns of Main-Chain Folding • The first common principle is that we can classify a protein based on the dominant secondary structural motifs • The second common principle is that many proteins are made up of more than one domain æ. - A domain is a compact, locally folded region of tertiary structure of roughly 150–250 amino acids. - Domains are interconnected by the polypeptide strand that runs through the whole molecule • A third common principle is that domains may themselves be composed of repeating secondary structure motifs, sometimes called “super secondary structures.”

• Concept: A protein domain is a compact, locally folded region of tertiary structure. Smaller proteins typically contain a single domain. Larger proteins may contain several domains.

• CATH (Class, Architecture, Topology, and “Homologous superfamily”) • This immense variation can be distilled down to four basic folding patterns in globular proteins: (1) those that are built about a packing of α helices, (2) those that are constructed on a framework of β sheets, (3) those that include both helices and sheets, and (4) those that contain little helix or sheet structure.

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Wednesday, January 31, 2018 • The Rossman fold is a common domain structure for an important class of enzymes that bind a nicotinamide adenine dinucleotide (NAD) cofactor and carry out reactions critical to the production of ATP in metabolic processes

• Concept The majority of globular protein structures can be broadly classified as: “mainly α,” “mainly β,” and “α + β.” A small number of globular proteins has little α or β secondary structure.

• Common principles of globular protein structure: - All globular proteins have a defined inside and outside - β sheets are usually twisted, or wrapped into barrel structures. - The polypeptide chain can turn corners in a number of ways, - Not all parts of globular proteins can be conveniently classified as helix, β sheet, or turns

6.4 | Factors Determining Secondary and Tertiary Structure - The Information for Protein Folding • Much evidence indicates that most of the information for determining the three-dimensional structure of a protein is carried in the amino acid sequence of that protein

• If we raise the temperature sufficiently, or make the pH extremely acidic or alkaline, or add to the solvent certain kinds of organic molecules like alcohols or urea, the protein structure will unfold

• Concept: Amino acid sequence (primary structure) determines secondary and tertiary structure. • Anfinsen’s work showed that a protein can self-assemble into its functional conformation, and it needs no information to guide it other than that contained in its amino acid sequence.

- The Thermodynamics of Folding • Since proteins fold spontaneously in buffer solutions that mimic the intracellular environment (pH ~ 7, 150 mM NaCl), the folding of a globular protein is clearly a thermodynamically favorable process under physiological conditions. In other words, the overall free energy change for folding must be negative

- Conformational Entropy • The folding process, which involves going from a multitude of “random-coil” conformations to a single folded structure, involves a decrease in randomness and thus a decrease in entropy (ΔS < 0). This change is termed the conformational entropy of folding:

- Random coil (higher entropy) → folded protein (lower entropy) • The conformational entropy change works against folding. • Concept: The decrease in conformational entropy when a protein folds disfavors folding. This is compensated in part by energy stabilization through internal noncovalent bonding.

- Charge-Charge Interactions • Charge–charge interactions can occur between positively and negatively charged side-chain groups

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Wednesday, January 31, 2018 • At neutral pH, one group will be charged positively and the other negatively, so an electrostatic attractive force exists between them

• Salt bridges • The mutual repulsion between pairs of the numerous similarly charged groups that are present in proteins in very acidic or basic solutions contributes further to the instability of the folded structure under these conditions

- Internal Hydrogen Bonds • Many of the amino acid side chains carry groups that are either good hydrogen bond donors or good acceptors. - Van der Waals Interactions • The weak induced dipole–induced dipole interactions between nonpolar groups can also make significant contributions to protein stability because in the folded protein, nonpolar groups are densely packed and thus make a large number of van der Waals contacts.

- The change in enthalpy for folding, ΔHU→F, is dominated by the differences in noncovalent bonding interactions between the unfolded and folded states:

• ΔHU→F → = Hfolded — Hunfolded • Typically, the only new covalent bonds that form upon folding are disulfide bonds (note, however, that most proteins do not contain disulfide bonds).

• Each individual noncovalent interaction can contribute only a small amount (at most only a few kilojoules per mole) to the overall negative enthalpy of interaction.

• Yet another factor makes a major contribution to the thermodynamic stability of many globular proteins. - When the chain folds into a globular structure, the hydrophobic residues become buried within the molecule (see Figure 6.19(b) æ), and the water molecules that were ordered around the solvent-exposed hydrophobic surfaces in the denatured protein are now released from the clathrates, thereby gaining freedom of motion. Thus, the randomness of the solvent is increased by internalizing hydrophobic groups via folding.

• ΔSuniverse = ΔSsystem + ΔSsurroundings • ΔSU →F = ΔSprotein + ΔSsolvent • ΔSprotein is the conformational entropy change for folding, which is negative; however, this unfavorable entropy change is counteracted by the favorable entropy increase for the solvent (Δ solvent ).

- In summary, the burial of hydrophobic surface area in the solvent-inaccessible core of the protein acts to stabilize the folded state by making the value of Δ → more positive.

- This source of protein stabilization is referred to as the hydrophobic effect • Concept: The burying of hydrophobic groups within a folded protein molecule produces a stabilizing entropy increase known as the hydrophobic effect.

• In summary, the stability of the folded structure of a globular protein depends on the interplay of three factors: - 1. The unfavorable conformational entropy change, which favors the unfolded state - 2. The favorable enthalpy contribution arising from intramolecular noncovalent interactions

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Wednesday, January 31, 2018 - 3. The favorable entropy change of the solvent arising from the burying of hydrophobic groups within the molecule

• On the whole, proteins are relatively unstable molecules. - The native, functional, conformations of many proteins can be perturbed easily by changes in intracellular conditions, such as increased temperature (~5 °C) or fluctuations in pH (typically in the range of +/- 2 pH units)

• This relatively low thermodynamic stability of proteins plays a role in the regulation of intracellular processes - The activity of certain proteins can be regulated by disrupting the native conformation - Disulfide Bonds and Protein Stability • Once folding has occurred, the three-dimensional structure is in some cases further stabilized by the formation of disulfide bonds between cysteine residues

• Concept Some folded proteins are stabilized by internal disulfide bonds, in addition to noncovalent forces. • Why don’t most proteins have disulfide bonds? - In fact, such bonds are relatively rare and are found primarily in proteins that are exported from cells, such as ribonuclease, BPTI, and insulin.

- One explanation is that the environment inside most cells is reducing and tends to keep sulfhydryl groups in the reduced state

- Prosthetic Groups, Ion-binding, and Protein Stability • The folded conformation of a protein may also be stabilized by the binding of a metal ion, as is the case for a class of DNA-binding proteins called zinc finger proteins (Figure 6.27(a) æ), or by binding to a cofactor or a prosthetic group æ (i.e., a small molecule that is required for the protein to be active).

• When the ion or cofactor is absent, the “stripped” protein is called an apoprotein æ, and when the ion or cofactor is bound, the resulting complex is called a holoprotein

6.5 | Dynamics of Globular Protein Structure - Kinetics of Protein Folding • The folding of globular proteins from their denatured conformations is a remarkably rapid process, often complete in less than a second under physiological conditions

• Levinthal’s paradox assumes that folding is a random process; thus, a protein must sample a vast number of possible conformations to find the desired native conformation

- One particularly well-studied intermediate is the so-called molten globule æ. The - molten globule is a compact, partially folded intermediate state that has native-like secondary structure and backbone folding topology, but lacks the defined tertiary structure interactions of the native state.

• Concept: Protein folding can be rapid but seems to involve well-defined intermediate states.

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Wednesday, January 31, 2018 • Concept: The molten globule is a compact, partially folded intermediate state that has native-like secondary structure and backbone folding topology, but lacks the defined tertiary structure interactions of the native state.

- The “Energy Landscape” Model of Protein Folding • Several proteins are known to fold via well-chara...