Chapter 4: The Three-Dimensional Structure of Proteins
Chapter 4: The Three-Dimensional Structure of Proteins
Chapter Outline
(4-1) Protein structure and function
(4-2) Primary structure of proteins
(4-3) Secondary structure of proteins
(4-4) Tertiary structure of proteins
(4-5) Quaternary structure of proteins
Protein Structure
Biologically active proteins are polymers consisting of amino acids linked by covalent peptide bonds.
Many conformations are possible for proteins due to the flexibility of amino acids around the \alpha-carbon bonds.
Native conformations refer to the single, stable, and functionally active 3-D shapes of proteins that exist at a state of lowest free energy under physiological conditions.
Many proteins exhibit large segments of random coil structure as they lack an obvious regular repeating structure.
Levels of Protein Structure
Primary structure (1°): The specific, linear order in which amino acids are covalently linked together by peptide bonds.
Read convention: from the N-terminal end (free amino group) to the C-terminal end (free carboxyl group).
Secondary structure (2°): The ordered 3-D arrangement, stabilized by hydrogen bonds, specifically involving the backbone atoms (\text{C═O} and \text{N—H} groups) of a polypeptide chain.
Tertiary structure (3°): The overall, complete 3-D arrangement of all atoms in a protein, including those in side chains (R groups) and any non-protein components (prosthetic groups).
Quaternary structure (4°): The arrangement and stoichiometry of multiple individual polypeptide chains (subunits) in relation to one another to form a functional complex.
Subunits: Individual polypeptide chains within a large, oligomeric protein.
Primary Structure
The amino acid sequence dictates the 3-D conformation of a protein, which in turn determines its properties and biological function.
Determining this sequence is a routine operation in classical biochemistry (e.g., Edman degradation).
Changes in one amino acid residue in a sequence (point mutation) can significantly alter biological functions.
Example: Substituting glutamic acid with valine at position \beta6 in hemoglobin causes sickle-cell anemia.
Secondary Structure
The secondary structure refers to the hydrogen-bonded arrangement of the polypeptide chain backbone.
Peptide chains are linked at opposite corners by swivels, allowing rotation around specific bonds.
Each amino acid residue has two bonds with free rotation, designated by the Ramachandran angles: phi (\Phi) and psi (\Psi).
\Phi: Bond between the \alpha-carbon (C_\alpha) and the amino nitrogen (N) of that residue.
\Psi: Bond between the \alpha-carbon (C_\alpha) and the carboxyl carbon (C') of that residue.
Steric hindrance restricts the possible values of \Phi and \Psi, visualized via a Ramachandran plot.
Types of secondary structures include:
\alpha-helix (common motif)
\beta-pleated sheet (extended structure)
Peptide Conformation
The peptide bond (\text{C═O} and \text{N—H}) exhibits partial double-bond character, making it rigid and planar (the amide plane).
The six atoms comprising the peptide group (C\alpha-C'-O and N-H-C\alpha) lie in a plane.
The groups defining the amide plane feature:
O
N
H
\alpha-Carbon
R side group
\alpha-Helix
The \alpha-helix is a rigid, rod-like structure stabilized by intra-chain hydrogen bonds running parallel to the helix axis.
The helical conformation permits a linear arrangement of peptide bond carbonyl (\text{C═O}) and amide (\text{N—H}) groups.
Features of the \alpha-helix:
It is predominantly a right-handed coil in proteins.
There are 3.6 residues for each complete turn of the helix.
The axial rise per residue (d) is approximately 1.5 ext{ Å} .
The linear distance between corresponding points on successive turns (pitch, P) is 5.4 ext{ Å} (P = 3.6 \times 1.5 ext{ Å}).
Each peptide bond is s-trans and planar.
The \text{C═O} group of each peptide bond forms a hydrogen bond with the \text{N—H} group of the fourth amino acid residue away (residue i bonds to residue i+4).
The \text{C═O} \cdots \text{H—N} hydrogen bonds are parallel to the helical axis.
All R groups project outward from the helix axis, minimizing steric interference.
Factors That Disrupt the \alpha-Helix
Disruption can occur due to:
Bending of the polypeptide backbone due to proline, known as a helix breaker. Proline's cyclic structure restricts rotation and results in the absence of the \text{N—H} group needed for crucial hydrogen bonding in the \alpha-amino group.
Strong electrostatic repulsion caused by proximity of several side chains bearing the same charges (e.g., clusters of Lys and Arg [$+$] or Glu and Asp [$-$]).
Steric repulsion or crowding caused by the proximity of bulky side chains (e.g., Val, Ile, and Thr) or \beta-branched amino acids.
\beta-Pleated Sheet
The \beta-pleated sheet involves interchain or intrachain hydrogen bonds between adjacent polypeptide segments (strands).
This gives rise to a characteristic zigzag or pleated structure where hydrogen bonds run perpendicular to the direction of the polypeptide chain.
Polypeptide chains lie adjacent to one another.
If adjacent strands run in the same N-terminal to C-terminal direction, the sheet is parallel; if they run in opposite directions, it is antiparallel. Antiparallel sheets tend to be more stable due to optimal linear hydrogen bond geometry.
R groups alternate above and below the plane of the sheet.
Each peptide bond is s-trans and planar.
Reverse Turns
Reverse turns (or \beta-turns) are compact segments where the polypeptide chain reverses direction abruptly, folding back on itself, often containing only four residues.
Reverse turns are critical for forming globular structures and are formed due to steric reasons, leading to a sudden change in direction of the polypeptide chain.
Glycine (minimal side chain) is commonly required or found at position 3 for Type II turns.
Proline is often the second residue in a reverse turn due to its fixed \Phi angle, providing the correct geometry for the turn.
Structures of Reverse Turns
Type I reverse turn: The side chain at residue 3 lies outside the loop, allowing nearly any amino acid residue at this position.
Type II reverse turn: The side chain of residue 3 is rotated 180^\circ and is positioned inside the loop, necessitating that the minimal side chain of glycine occupies this position to avoid steric conflict.
Supersecondary Structures and Domains
Supersecondary structures (or motifs) result from the specific combination of \alpha-helices and \beta-strands linked by loops or turns.
\beta\alpha\beta unit: Two parallel strands of \beta-sheet connected by a stretch of \alpha-helix.
\alpha\alpha unit: Contains two antiparallel \alpha-helices packed together, often referred to as helix-turn-helix, frequently found in DNA-binding proteins.
\beta-meander: An antiparallel sheet formed by tight reverse turns connecting stretches of polypeptide chain.
Greek key: A motif formed when four adjacent antiparallel strands fold into a specific pattern, resembling a Greek ornamental pattern.
Domains are larger, independent folding units within a single polypeptide chain, typically consisting of 50 to 350 amino acid residues.
The \beta-barrel is created when extensive \beta-sheets fold back on themselves, often forming a pore. Domains provide insights into protein folding mechanics and structure, and similar domain conformations are associated with similar functional roles.
Fibrous Proteins
Fibrous proteins consist of polypeptide chains organized approximately parallel along a single axis, forming repetitive units.
They form long fibers or sheets that are highly resistant to stretching, mechanically strong, insoluble in water, and play important structural roles in nature.
Examples include:
\alpha-Keratin in hair, wool, nails (composed primarily of \alpha-helices twisted together).
Collagen in connective tissues, including cartilage, bones, and skin (characterized by a unique triple helix structure).
Globular Proteins
Globular proteins have a complex 3-D backbone folding on itself to produce a compact, roughly spherical shape.
Most polar side chains are situated on the protein surface (outside), allowing interaction with the aqueous environment via hydrogen bonding and ion-dipole interactions.
Nonpolar side chains are generally buried in the interior (hydrophobic core).
They contain significant and varied sections of \alpha-helix and \beta-sheet; overall, they maintain compact structures and are highly soluble in water and salt solutions.
Tertiary Structure of Proteins
The tertiary structure represents the definitive 3-D arrangement of all atoms, including the conformations of side chains and the spatial location of any prosthetic groups.
In contrast to fibrous proteins where backbones are extended, in globular proteins, tertiary structure dictates how distinct secondary structural elements (helical and pleated-sheet sections) fold and pack tightly back on one another.
Interactions between side chains (R groups) are the critical stabilizing forces for tertiary folding.
Forces Involved in Tertiary Structures
The stability of tertiary structures depends primarily on various noncovalent interactions and one major covalent bond:
Hydrophobic interactions: The primary driving force for protein folding, causing nonpolar side chains to aggregate internally away from water, increasing the entropy of the surrounding water molecules.
Hydrogen bonding between polar side chains of amino acids (e.g., Ser and Thr side chains).
Electrostatic attraction (or salt bridges) between oppositely charged side chains (e.g., terminal Lys/Arg and Asp/Glu groups: $\text{—}\text{NH}_3^+ \cdots \text{—}\text{COO}^-$).
van der Waals forces (weak, short-range interactions) contributing to the dense packing of the protein interior.
Complexation of several side chains with a single metal ion (coordinate covalent bonds).
Disulfide bonds ($\text{—}\text{S}\text{—}\text{S}\text{—}$) formed by the oxidation of two cysteine side chains ($\text{—}\text{SH}$). These covalent cross-links significantly restrict folding patterns and increase stability.
Disulfide bonds are present in some secreted or stable enzymes like chymotrypsin and trypsin, but absent in typical intracellular proteins like myoglobin and hemoglobin.
Myoglobin
Myoglobin is a relatively small, globular protein consisting of a single polypeptide chain of 153 amino acid residues and a non-protein prosthetic group called heme located in a deeply buried hydrophobic pocket.
Heme: An iron-containing cyclic compound essential for oxygen binding, giving myoglobin its characteristic compact structure. The polypeptide chain is richly comprised of eight major \alpha-helical regions (A through H), stabilized by extensive backbone hydrogen bonding.
It completely lacks any \beta-pleated sheet regions.
The exterior hosts most hydrophilic polar side chains while nonpolar chains and specific histidine residues (proximal and distal) interact with the heme group and bind oxygen inside.
Structure of Myoglobin
Myoglobin features a heme group (\text{Fe}^{2+}) embedded in its structure, surrounded by specific \alpha-helical regions denoted by letters (e.g., FG, H, F, COO-, CD, E, D, B, H, AB, EF, NH, GH).
Heme Group
The presence and conformation of the heme group profoundly affect the overall structure and function of the polypeptide (tertiary structure).
Heme structure:
Consists of a central metal ion \text{Fe}(\text{II}) (ferrous iron) that requires six coordination sites (ligands).
Four sites: Occupied by nitrogen (N) atoms from the four pyrrole-type rings (in the porphyrin structure).
Fifth site: Occupied by the nitrogen atom of the imidazole side chain from Histidine F8 (the proximal histidine), covalently linking the protein to the heme.
Sixth site: The binding site for gaseous ligands, notably \text{O}_2 .
The large organic part is protoporphyrin IX, made of four five-membered pyrrole rings linked by methine ($\text{—}\text{CH═}$) groups.
Oxygen: Imperfect Binding to the Heme Group
Affinity of free heme for dangerous ligands is extremely high; specifically, the affinity of free heme for carbon monoxide (\text{CO}) is approximately 25,000 times higher than for oxygen (\text{O}_2).
In myoglobin, the presence of the Distal Histidine (His E7) sterically hinders linear binding of CO, reducing its effective affinity compared to free heme and favoring the physiologically correct angled binding of \text{O}_2.
Oxygen and Carbon Monoxide Binding to the Heme Group of Myoglobin
The binding orientations of \text{O}_2 and \text{CO} to the heme group of myoglobin are controlled by the specific positioning of two histidine residues: His E7 (Distal Histidine, which interacts with the ligand but doesn't bind to \text{Fe}^{2+}) and His F8 (Proximal Histidine, which binds directly to \text{Fe}^{2+}).
Denaturation and Refolding
Denaturation refers to the process of unraveling the ordered native 3-D structure of a macromolecule caused by the specific breakdown of noncovalent stabilizing interactions (and potentially disulfide bonds). This results in loss of biological function.
Denaturation can occur due to:
Heat: Increases kinetic energy, disrupting weak noncovalent interactions, especially hydrophobic interactions.
Large changes in pH: Alters the ionization state (charges) of acidic and basic side chains, disrupting electrostatic interactions (salt bridges) and hydrogen bonds.
Detergents (e.g., sodium dodecyl sulfate [SDS]): Amphipathic molecules that coat and penetrate the protein structure, disrupting the internal hydrophobic core and forcing the protein to linearize.
Chaotropic agents (e.g., Urea and guanidine hydrochloride): Highly soluble in water, they disrupt the structure of water and competitively form hydrogen bonds with the protein backbone and side chains, breaking internal protein-protein H-bonds.
Reducing agents (e.g., \beta-mercaptoethanol): Used to cleave stabilizing disulfide bonds ($\text{—}\text{S}\text{—}\text{S}\text{—} \rightarrow 2 \text{—}\text{SH}$).
Quaternary Structure of Proteins
The quaternary structure describes proteins consisting of more than one polypeptide chain associated noncovalently.
Each constituent chain is referred to as a subunit or protomer.
Oligomers are proteins with a small number of subunits, including dimers, trimers, and tetramers.
Chains interact noncovalently through the same forces stabilizing tertiary structure: electrostatic attractions, hydrogen bonds, and especially hydrophobic interactions.
Allosteric describes the critical property of multisubunit proteins where the binding of a ligand or a conformational change in one subunit significantly alters the conformation and binding affinity of another subunit.
Hemoglobin
Hemoglobin (\text{Hb}), the primary oxygen transport protein in red blood cells, is a functional tetramer comprised of four subunits:
Two identical \alpha-chains (each 141 residues long).
Two identical \beta-chains (each 146 residues long).
The overall structure is denoted as \alpha2\beta2, and each subunit is structurally very similar to myoglobin.
Most amino acids of the \alpha-chain, \beta-chain, and myoglobin are homologous (share sequence and structural similarity).
Oxygen Binding of Hemoglobin (Hb)
Unlike myoglobin, which binds a maximum of one \text{O}2 molecule, hemoglobin can bind up to four molecules of \text{O}2 (one per heme group in each of the four subunits).
The binding of \text{O}2 to hemoglobin exhibits positive cooperativity, meaning that the binding of the first oxygen molecule causes a conformational shift that increases the affinity of the remaining subunits for subsequent \text{O}2 molecules.
Myoglobin versus Hemoglobin
Myoglobin (M):
Function: Oxygen storage in muscle tissue, ready for emergency use.
Requirement: Exhibits a hyperbolic binding curve. Needs strong binding to \text{O}2 at very low partial pressures found in muscle, achieving 50\% saturation (P{50}) at approximately 1 ext{ torr} partial pressure of \text{O}_2.
Hemoglobin (Hb):
Function: Oxygen transport from lungs to peripheral tissues.
Requirement: Exhibits a sigmoidal (S-shaped) binding curve characteristic of cooperative systems. Needs strong binding in the lungs (high \text{O}2 pressure) and easy release in the tissues (low \text{O}2 pressure). Achieves near 100\% saturation at 100 ext{ torr} (\text{p} \text{O}_2 in the lungs).
Structures of Hemoglobin
Hemoglobin exists primarily in two quaternary conformations:
Tensed (T) state: The low-affinity, deoxygenated form (\text{deoxyHb}). Stabilized by salt bridges between subunits.
Relaxed (R) state: The high-affinity, oxygenated form (\text{oxyHb}). Oxygen binding shifts the equilibrium from T to R, breaking inter-subunit salt bridges.
Conformational Changes That Accompany Hemoglobin Function
Ligands involved in cooperative effects (allosteric effectors), such as protons (\text{H}^+) and carbon dioxide (\text{CO}2), impact hemoglobin's affinity for \text{O}2 by altering its quaternary structure:
The Bohr effect refers to the decrease in \text{O}2 affinity of hemoglobin with increasing acidity (decreasing pH, or increasing [\text{H}^+]). Tissues undergoing high metabolic activity release \text{H}^+ and \text{CO}2, prompting \text{Hb} to release \text{O}_2.
Oxygenated hemoglobin acts as a stronger acid (due to conformational changes releasing protons) since it has a lower \text{p}K_a than the deoxygenated form, causing greater \text{H}^+ affinity in the deoxygenated state (T state).
Oxygen Saturation Curves for Myoglobin and Hemoglobin at Different pH Values
Oxygen saturation curves graphically illustrate how the pH affects the ability of myoglobin and hemoglobin to bind oxygen across a range of partial pressures of \text{O}_2.
The \text{Hb} curve shifts right (lower affinity, increased \text{O}_2 release) as pH decreases, highlighting the Bohr effect. The myoglobin curve remains largely unaffected by physiological pH changes.
Conformational Changes That Accompany Hemoglobin Function
Hemoglobin binding is also regulated by an important allosteric effector found in red blood cells: 2,3-bisphosphoglycerate (\text{BPG}).
\text{BPG} binds preferentially and tightly to a central cavity in the Tensed (T) state of deoxyhemoglobin.
Binding is stabilized via electrostatic interactions with positively charged amino acid side chains (Lys, His, N-terminal amines) lining the central cavity.
\text{BPG} lowers the oxygen affinity of \text{Hb}. In the absence of \text{BPG}, the oxygen-binding capacity of hemoglobin would be too high to effectively release \text{O}2 in peripheral tissues. \text{BPG} concentration is regulated to ensure efficient \text{O}2 supply, including compensating for high altitude or supplying \text{O}_2 to the growing fetus (fetal \text{Hb} binds \text{BPG} less tightly).
Binding of BPG to Deoxyhemoglobin
The binding involves highly specific noncovalent interactions in the central pocket (cavity) of the tetramer, which is only open in the T state. The presence of \text{BPG} stabilizes the T state, making it harder for \text{O}2 to bind and shift it to the R state, thereby facilitating \text{O}2 dissociation in the tissues.