Protein Structure and Folding Notes

Four Levels of Protein Structure

• Protein molecules adopt a specific three-dimensional conformation.
• This structure enables the fulfillment of a specific biological function.
• This structure is called the native fold.
• Protein structures are not static; they undergo conformational changes ranging from subtle to dramatic.

Stabilization of Protein Conformation

• Protein conformation is stabilized largely by weak interactions.
• Chemical interactions that counteract entropy and lead to folding and stabilize the native conformation include:
  • Disulfide (covalent) bonds
  • Weak (noncovalent) interactions and forces:
    • Hydrogen bonds
    • Hydrophobic effect
    • Ionic interactions

Primary Structure: The Peptide Bond

• The peptide bond is a resonance hybrid of two structures.
• Resonance causes the peptide bonds:
  • To be quite rigid and planar.
  • To exhibit a large dipole moment in the favored trans configuration.
• The peptide bond has partial double bond character.
• Early X-ray crystal structures showed the peptide C—N bond is somewhat shorter than the C—N bond in a simple amine.

Characteristics of the Peptide Bond

• The peptide bond can always be considered to be in the trans conformation due to steric hindrance in the cis conformation.
• The rigid peptide plane only allows free rotation around the N—Cα and Cα—C bonds.
  • Rotation around the peptide bond is not permitted due to resonance structure.
  • Rotation around bonds connected to the α carbon is permitted.
    • $\phi$ (phi): angle around the α carbon—amide nitrogen bond
    • $\psi$ (psi): angle around the α carbon—carbonyl carbon bond
• The organization around the peptide bonds determines the secondary structure of the protein.

Dihedral Angles

• A dihedral angle is the angle at the intersection of two planes.
  • $\phi$ involves N—Cα bond, and $\psi$ involves the Cα—C bond.
  • Both $\phi$ and $\psi$ are defined as $\pm 180^\circ$ when the polypeptide is fully extended and all peptide groups are in the same plane
• Many $\phi$ and $\psi$ combinations are unfavorable because of steric crowding of backbone or sidechain atoms.
• Some $\phi$ and $\psi$ combinations are more favorable due to H-bonding interactions along the backbone.

Ramachandran Plot

• A Ramachandran plot shows the distribution of $\phi$ and $\psi$ angles corresponding to common secondary structures.

Secondary Structure

• Secondary structure refers to a local spatial arrangement of the polypeptide backbone.
• Two regular arrangements are common:
  • The $\alpha$ helix – stabilized by hydrogen bonds between nearby residues.
  • The $\beta$ sheet – stabilized by hydrogen bonds between adjacent segments that may not be close to each other in the primary sequence.

Idealized $\phi$ and $\psi$ Angles for Common Secondary Structures in Proteins

• $\alpha$ Helix: $\phi = -57^\circ$, $\psi = -47^\circ$
• $\beta$ Conformation:
  • Antiparallel: $\phi = -139^\circ$, $\psi = +135^\circ$
  • Parallel: $\phi = -119^\circ$, $\psi = +113^\circ$
• Collagen triple helix: $\phi = -51^\circ$, $\psi = +153^\circ$
• $\beta$ Turn type I
  • i + 1: $\phi = -60^\circ$, $\psi = -30^\circ$
  • i + 2: $\phi = -90^\circ$, $\psi = 0^\circ$
• $\beta$ Turn type II
  • i + 1: $\phi = -60^\circ$, $\psi = +120^\circ$
  • i + 2: $\phi = +80^\circ$, $\psi = 0^\circ$

The $\alpha$ Helix

• Helical backbone is held together by hydrogen bonds between the backbone amides of an n and n + 4 amino acids.
• Regularly repeating: It is a right-handed helix with 3.6 residues (5.4 Å) per turn.
• Side chains point outward and are roughly perpendicular with the helical axis and don’t contribute to secondary structure.

Energetics of the $\alpha$ Helix

• The $\alpha$ Helix is energetically favorable due to extensive hydrogen bonding
• Every C−O hydrogen bonded to N−H 4 residues away
• Amino acids 1 and 4 are on the same side/face of an alpha helix
• Amino acids 3 and 4 residues away from each other may function together on one “face” of the helix

Sequence Affects Helix Stability

• Proline (Pro) acts as a helix breaker because the rotation around the N-Cα ($\phi$-angle) bond is impossible. Also, there is no substituent amide hydrogen to participate in hydrogen bonds with other residues
• Glycine (Gly) acts as a helix breaker because the tiny R group supports other conformations (unconstrained and adopting “disallowed” angles)

Propensity of Amino Acid Residues to Take Up an α-Helical Conformation

• Larger $\Delta\Delta G^\circ$ values reflect greater difficulty taking up the $\alpha$-helical structure compared to Alanine which is 0.

Electrostatic Repulsion in Alpha-Helices

• Negatively charged carboxyl groups of adjacent Glutamate and/or Aspartate residues repel each other so strongly that they prevent formation of the $\alpha$ helix.
• For the same reason, adjacent Lysine and/or Arginine residues, with positively charged R groups will also repel each other and prevent formation of the $\alpha$ helix.
• Adjacent amino acids with side chains of the same charge are not favored in alpha-helices.

$\beta$ Sheets

• The planarity of the peptide bond and tetrahedral geometry of the α carbon create a pleated sheet-like structure.
• Sheet-like arrangement of the backbone is held together by hydrogen bonds between the backbone amides in different strands.
• Side chains protrude from the sheet, alternating in an up-and-down direction.

Parallel and Antiparallel $\beta$ Sheets

• Multi $\beta$-strand interactions are called sheets.
• Sheets are held together by the hydrogen bonding of amide and carbonyl groups of the peptide bond from opposite strands.
• Two major orientations of $\beta$ sheets are determined by the directionality of the strands within:
  • Parallel sheets have strands that are oriented in the same direction.
  • Antiparallel sheets have strands that are oriented in opposite directions.

Hydrogen Bonding in $\beta$ Sheets

• In parallel $\beta$ sheets, the H-bonded strands run in the same direction with bent (weaker) hydrogen bonds.
• In antiparallel $\beta$ sheets, the H-bonded strands run in opposite directions with linear (stronger) hydrogen bonds.

$\beta$ Turns

• $\beta$ turns occur frequently whenever strands in $\beta$ sheets change direction.
• The 180° turn is accomplished over four amino acids.
• The turn is stabilized by a hydrogen bond from a carbonyl oxygen to amide proton three residues down the sequence.
• Proline in position 2 or glycine in position 3 are common in $\beta$ turns.

Proline Isomers

• Most peptide bonds not involving proline are in the trans configuration (>99.95%).
• For peptide bonds involving proline, about 6% are in the cis configuration. Most of this 6% involve $\beta$ turns.
• Proline isomerization is catalyzed by proline isomerases.

Protein Tertiary Structure

• Tertiary structure refers to the overall spatial arrangement of atoms in a protein.
• The native fold is stabilized/ordered against entropy by a large number of favorable interactions (mostly weak) within the protein
• The conformations existing under a given set of conditions are usually the ones that are thermodynamically the most stable—that is, having the lowest Gibbs free energy (G).

Techniques for Determining Tertiary Structures of Proteins

• X-Ray Diffraction (X-ray crystallography)
  • The spacing of atoms in a crystal lattice can be determined by measuring the locations and intensities of spots produced on photographic film by a beam of x-rays of given wavelength, after the beam has been diffracted by the electrons of the atoms.
• Nuclear Magnetic Resonance
  • An advantage of nuclear magnetic resonance (NMR) studies is that they are carried out on macromolecules in solution, whereas x-ray crystallography is limited to molecules that can be crystallized.

Favorable Interactions in Proteins

• Hydrophobic effect
  • The release of water molecules from the structured solvation layer around the molecule as protein folds increases the net entropy.
• Hydrogen bonds
  • Interaction of N−H and C=O of the peptide bond leads to local regular structures such as $\alpha$ helices and $\beta$ sheets.
• London dispersion (Van der Waals interactions)
  • Medium-range weak attraction between all atoms contributes significantly to the stability in the interior of the protein.
• Electrostatic interactions
  • Long-range strong interactions between permanently charged groups.
  • Salt bridges, especially those buried in the hydrophobic environment, strongly stabilize the protein.
• Proteins can also be stabilized by disulfide bonds between cysteines

Strength of Interactions in Proteins

• 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.
• Of the weak interactions, the ”hydrophobic effect” generally predominates as a force in protein folding
• the interior of a structured protein is generally a densely packed core of hydrophobic amino acid side chains.

Entropy and Protein Folding

• Increase in entropy of water is the major thermodynamic driving force for the association of hydrophobic groups in aqueous solution.
• 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.
• In this way, hydrogen bonds often have an important role in guiding the protein-folding process. Cooperativity of hydrogen bonding helps drive protein folding

Hydrogen Bonding and Ionic Interactions

• The presence of hydrogen/ionic bonding groups without partners in the hydrophobic core of a protein can be so destabilizing that conformations containing these groups are often thermodynamically untenable.
• Any polar or charged groups in the protein interior must have suitable partners for hydrogen bonding or ionic interactions.

Salt Bridges and Dielectric Constant

• The strength of a salt bridge increases as it moves to an environment of lower dielectric constant, $\epsilon$: from the polar aqueous solvent ($\epsilon$ near 80) to the nonpolar protein interior ($\epsilon$ near 4) because it has less potential interaction partners like water.
• Salt bridges, especially those that are partly or entirely buried, can provide significant stabilization to a protein structure.

Van der Waals Interactions

• Van der Waals effects (London dispersion forces) in protein folding
• Random variations in the positions of the electrons around one nucleus may create a transient electric dipole, which induces a transient, opposite electric dipole in the nearby atom.
• The two dipoles weakly attract each other, bringing the two nuclei closer.

Disulfide Bonding in Tertiary Structure

• The environment within most cells is highly reducing due to high concentrations of reductants such as glutathione, and most sulfhydryls will thus remain in the reduced state.
• In eukaryotes, disulfide bonds are found primarily in secreted, extracellular proteins (for example, the hormone insulin).

Fibrous and Globular Proteins

• Two major groups into which many proteins can be classified are fibrous and globular
• Fibrous proteins, with polypeptide chains arranged in long strands or sheets (largely of a single type of secondary structure, and their tertiary structure is relatively simple) provide support, shape, and external protection
• Globular proteins, with polypeptide chains folded into a spherical or globular shape often contain several types of secondary structure. Most enzymes and regulatory proteins are globular proteins (e.g. Collagen and myoglobin)

Fibrous Proteins: From Structure to Function

• $\alpha$ Helix, cross-linked by disulfide bonds:
  • Tough, insoluble protective structures of varying hardness and flexibility.
  • Examples: $\alpha$-Keratin of hair, feathers, nails
• $\beta$ Conformation:
  • Soft, flexible filaments.
  • Example: Silk fibroin
• Collagen triple helix:
  • High tensile strength, without stretch.
  • Example: Collagen of tendons, bone matrix

Collagen Structure

• The repeating tripeptide sequence Gly–X–Pro or Gly–X–4-Hyp adopts a helical structure with three residues per turn.
• Gly (shown in red) because of its small size, is required at the tight junction where the three chains are in contact
• Many triple-helices assemble into a collagen fibril.

4-Hydroxyproline in Collagen

• Forces the proline ring into a favorable pucker
• Offers more hydrogen bonds between the three strands of collagen
• The posttranslational processing is catalyzed by prolyl hydroxylase and requires $\alpha$-ketoglutarate, molecular oxygen, and ascorbate (vitamin C).

Collagen Fibrils

• Collagen superstructures are formed by cross-linking of collagen triple-helices to form collagen fibrils.
• Crosslinks are covalent bonds between Lys or HyLys, or His amino acid residues.

Scurvy

• Scurvy is a condition caused by a lack of vitamin C in the diet, resulting in a lack of hydroxyproline and defective collagen

α-Keratin Structure

• Super-twisting of two alpha-helical strands amplifies the strength of the overall structure, just as strands are twisted to make a strong rope.
• The surfaces where the two α helices touch are made up of hydrophobic amino acid residues, their R groups meshed together in a regular interlocking pattern.
• This permits a close packing of the polypeptide chains within the left-handed supertwist.
• Not surprisingly, α-keratin is rich in the hydrophobic residues Ala, Val, Leu, Ile, Met, and Phe.

Silk Fibroin

• Fibroin is the main protein in silk from moths and spiders.
• Antiparallel $\beta$ sheet structure
• Small side chains (Ala and Gly) allow the close packing of sheets.
• Structure is stabilized by hydrogen bonding within sheets

Motifs (folds)

• Specific arrangement of several secondary structure elements; all $\alpha$ helix, all $\beta$ sheet, mixture of both
• Globular proteins are composed of different motifs folded together.
• A motif or fold is a recognizable folding pattern involving two or more elements of secondary structure and the connection(s) between them

Intrinsically Disordered Proteins

• Contain protein segments that lack definable structure
• Intrinsically disordered proteins regions have properties that are distinct from those of classical, structured proteins. They lack a hydrophobic core and instead are characterized by high densities of charged amino acid residues such as Lys, Arg, and Glu. Pro residues are also prominent, as they tend to disrupt ordered structures.
• The lack of an ordered structure can facilitate a kind of functional promiscuity, allowing one protein to interact with multiple partners.

Quaternary Structure

• A quaternary structure is formed by the assembly of individual polypeptides into a larger functional cluster.

Protein Stability and Folding

• A protein’s function depends on its 3D structure.
• Loss of structural integrity with accompanying loss of activity is called denaturation.
• Proteins can be denatured by:
  • heat or cold
  • pH extremes
  • organic solvents

Ribonuclease Refolding Experiment

• Ribonuclease contains eight cysteines linked via four disulfide bonds.
• Urea in the presence of 2-mercaptoethanol fully denatures ribonuclease.
• When urea and 2-mercaptoethanol are removed, the protein spontaneously refolds, and the correct disulfide bonds are reformed.
• The sequence alone determines the native conformation.
• Mercaptoethanol (HOCH2CH2SH) reduces and thus cleaves the disulfide bonds
• Renaturation involves reestablishing the correct disulfide cross-links.

Proteins Folding Follow a Distinct Path

• Amino acid sequence of a 56-residue peptide

Levinthal’s Paradox

• Proteins fold to the lowest-energy fold in the microsecond to second time scales. How can they find the right fold so fast?
• It is mathematically impossible for protein folding to occur by randomly trying every conformation until the lowest-energy one is found (Levinthal’s paradox).
• Search for the minimum is not random because the direction toward the native structure is thermodynamically most favorable.

Proteostasis

• The continual maintenance of the active set of cellular proteins required under a given set of conditions is called proteostasis
• Maintenance of cellular protein activity is accomplished by the coordination of many different pathways.

Chaperones

• The chaperones do not actively promote the folding of the substrate protein, but instead prevent aggregation of unfolded peptides, Giving them a chance to find the correct folds

Protein Misfolding

• Native (correctly folded) $\beta$ amyloid is a soluble globular protein,
• Misfolded $\beta$ amyloid promotes aggregation at newly exposed protein-protein interface.
• Before folding is complete, the $\beta$-sheet regions of one polypeptide associate with the same region in another polypeptide, forming the nucleus of an amyloid. Additional protein molecules slowly associate with the amyloid and extend it to form a fibril (Alzheimer’s)