chapter 2 Protein Structure and Properties

Polypeptide Chains and Peptide Bond Structure

• Polypeptide Polarity and Directionality:

  • Polypeptides are synthesized and named from the N-terminus (amino-terminal residue) to the C-terminus (carboxyl-terminal residue).

  • The amino-terminal residue possesses a free or protonated amino group ($\text{}^+\text{H}_3\text{N}-$), while the carboxyl-terminal residue possesses a free or ionized carboxylate group ($\text{-COO}^-$).

  • Example pentapeptide sequence structure: Tyrosine ($\text{Tyr}$) - Glycine ($\text{Gly}$) - Glycine ($\text{Gly}$) - Phenylalanine ($\text{Phe}$) - Leucine ($\text{Leu}$).

• Chemical Characteristics of the Peptide Bond:

  • The peptide bond links the carbonyl carbon ($\text{C=O}$) of one amino acid residue to the amide nitrogen ($\text{N-H}$) of the adjacent residue ($\text{C-N}$ bond).

  • Resonance Structures:

    • The peptide bond exists as a resonance hybrid between a uncharged single-bond structure ($\text{C-N}$ with a double-bonded oxygen $\text{C=O}$) and a dipolar structure containing a double bond between carbon and nitrogen ($\text{C=N}^+$) and a single-bonded negatively charged oxygen ($\text{C-O}^-$).

    • This resonance gives the $\text{C-N}$ bond partial double-bond character (~$40\%$ double-bond character), restricting rotation around the backbone peptide bond.

• Planar Peptide Unit:

  • Due to resonance, six atoms lie in a single rigid plane: the alpha-carbon ($\text{C}_{\alpha1}$), carbonyl carbon ($\text{C}$), carbonyl oxygen ($\text{O}$), amide nitrogen ($\text{N}$), amide hydrogen ($\text{H}$), and the next alpha-carbon ($\text{C}_{\alpha2}$).

Torsion Angles and Conformational Flexibility

• Definition of Dihedral / Torsion Angles:

  • Protein backbone folding is governed by rotation around two single bonds per amino acid residue:

    • $\phi$ (phi): The torsion angle of rotation around the $\text{N-C}_{\alpha}$ bond (defined by atoms $\text{C-N-C}_{\alpha}\text{-C}$).

    • $\psi$ (psi): The torsion angle of rotation around the $\text{C}_{\alpha}\text{-C}$ bond (defined by atoms $\text{N-C}_{\alpha}\text{-C-N}$).

    • $\omega$ (omega): The torsion angle around the $\text{C-N}$ peptide bond, which is typically restricted to $180^\circ$ (trans conformation) or $0^\circ$ (cis conformation) due to resonance planar rigidity.

• Specific Angle Conformations:

  • A conformation with $\phi = -80^\circ$ represents a specific rotational orientation about the $\text{N-C}_{\alpha}$ bond.

  • A conformation with $\psi = +85^\circ$ represents a specific rotational orientation about the $\text{C}_{\alpha}\text{-C}$ bond.

• Steric Hindrance and Disfavored Conformations:

  • Not all combinations of $\phi$ and $\psi$ are physically possible because many result in steric collisions (clashes) between backbone atoms or sidechain atoms.

  • Example disfavored conformation: $\phi = +90^\circ$, $\psi = -90^\circ$, where the carbonyl oxygen atom collides sterically with the sidechain group or adjacent backbone hydrogens.

• Ramachandran Plot:

  • A Ramachandran plot maps all theoretical values of $\psi$ (y-axis, $-180^\circ$ to $+180^\circ$) versus $\phi$ (x-axis, $-180^\circ$ to $+180^\circ$).

  • Sterically allowed/favored regions appear as distinct clusters corresponding to regular secondary structural motifs:

    • Upper-left quadrant: $\beta$-strands and $\beta$-sheets.

    • Lower-left quadrant: Right-handed $\alpha$-helices.

    • Upper-right quadrant: Rare left-handed $\alpha$-helices.

Levels of Protein Structure

• Structural Hierarchy:

  • Primary Structure ($1^\circ$): The specific, linear amino acid sequence joined by covalent peptide bonds (e.g., Leu-Tyr-Gln-Leu-Glu-Asn-Tyr).

  • Secondary Structure ($2^\circ$): Local spatial arrangement of the polypeptide backbone into regular recurring structures ($\alpha$-helices, $\beta$-strands, turns) stabilized by backbone hydrogen bonds.

  • Tertiary Structure ($3^\circ$): Three-dimensional folding of a single polypeptide chain into compact globular or fibrous units (domains).

  • Quaternary Structure ($4^\circ$): Spatial arrangement and non-covalent or covalent interaction of multiple individual polypeptide subunits in a multi-protein complex.

Structure-Function Relationships and Macromolecular Assemblies

• Functional Structures:

  • DNA Polymerase: Forms a circular, clamp-like ring structure surrounding double-stranded DNA to execute high-fidelity replication along the nucleic acid template strand.

• Supramolecular Muscle Assembly:

  • Single Muscle Fiber (Cell): Encased by a plasma membrane, containing multiple nuclei and densely packed bundle of myofibrils.

  • Myofibrils and Sarcomere Organization:

    • A single myofibril consists of repeating contractile structural units termed sarcomeres.

    • Sarcomere Boundaries: Bounded by two dark Z lines.

    • I Band: Light region containing thin filaments (actin) anchored to the Z line.

    • A Band: Dark central region encompassing the entire length of thick filaments (myosin).

    • H Zone: Central lighter area within the A band containing thick filaments only.

    • Filaments: Interdigitating thick filaments (myosin) and thin filaments (actin) slide past one another during contraction.

• Protein Flexibility and Domain Motion:

  • Lactoferrin: Demonstrates large-scale conformational changes upon ligand binding.

  • In the iron-free (apo) state, two globular domains are held open.

  • Upon binding an iron atom ($\text{Fe}$), the two domains pivot around a central hinge region to enclose the iron ion securely inside the binding pocket.

• Protein Structure Determination via X-Ray Crystallography:

  • Crystalline State: Purified proteins are crystallized into ordered, repeating three-dimensional arrays.

  • X-Ray Diffraction: A focused X-ray beam bombards the protein crystal, scattering rays to yield a distinct diffraction pattern registered on detector plates.

  • Pattern Processing: Mathematical conversion (Fourier transform) of spot intensities and positions reveals electron density maps used to determine precise atomic coordinates.

Types of Secondary Structure

The $\alpha$-Helix

• Structural Definition and Hydrogen Bonding Pattern:

  • The $\alpha$-helix is a tightly coiled, rod-like backbone structure.

  • From the N-terminus to C-terminus, a hydrogen bond forms between the carbonyl oxygen ($\text{C=O}$) of amino acid residue $i$ and the amide nitrogen hydrogen ($\text{N-H}$) of amino acid residue $i+4$.

  • This repeated $\text{C=O}_i \cdots \text{H-N}_{i+4}$ hydrogen bonding pattern stabilizes the helical framework along the longitudinal axis.

• Helix Parameters and Physical Characteristics:

  • Residues per turn: Approximately $3.6$ amino acid residues per complete $360^\circ$ turn.

  • Torsion Angles: Right-handed $\alpha$-helices occupy a specific favored region on the Ramachandran plot centered at $\phi = -57^\circ$ and $\psi = -47^\circ$.

  • Handedness: Right-handed $\alpha$-helices are common and favorable in proteins; left-handed $\alpha$-helices are energetically unfavorable and extremely rare.

• Hydrogen Bond Energy and Geometry:

  • Standard backbone hydrogen bond energy is approximately $1.5\,\text{kcal/mol}$.

  • Hydrogen bond distance parameters: $\text{O}\cdots\text{O}$ interatomic distance is $2.8\,\text{\AA}$ ($0.28\,\text{nm}$); $\text{H}\cdots\text{O}$ distance is $1.8\,\text{\AA}$ ($0.18\,\text{nm}$); covalent $\text{N-H}$ distance is $1.0\,\text{\AA}$.

  • Optimal linear hydrogen bond alignment occurs at an angle near $180^\circ$ between donor and acceptor atoms.

• Amphipathic $\alpha$-Helices and Helical Wheel Projections:

  • Sidechains project outward and downward from the helical mainchain core.

  • When plotted on a 2D helical wheel projection (viewed looking down the central axis), amino acids separated by 3 to 4 residues lie on the same side of the helix.

  • Example 18-residue sequence: 1-MLQSMVSLLQSLVSLIIQ-18:

    • Nonpolar / Hydrophobic face (colored yellow): $1\text{-Met}$, $2\text{-Leu}$, $5\text{-Met}$, $6\text{-Val}$, $8\text{-Leu}$, $9\text{-Leu}$, $12\text{-Leu}$, $13\text{-Val}$, $15\text{-Leu}$, $16\text{-Ile}$, $17\text{-Ile}$.

    • Polar / Uncharged face (colored green): $3\text{-Gln}$, $4\text{-Ser}$, $7\text{-Ser}$, $10\text{-Gln}$, $11\text{-Ser}$, $14\text{-Ser}$, $18\text{-Gln}$.

• Three Elements of $\alpha$-Helix Stabilization:

  1. Intramolecular backbone hydrogen bonds ($\text{C=O}_i \cdots \text{H-N}_{i+4}$).

  2. Favorable backbone torsion angles ($\phi = -57^\circ$, $\psi = -47^\circ$) that minimize steric clashes between sidechain atoms and backbone atoms.

  3. Hydrophobic interactions between nonpolar sidechains sequestered on one face of the helical surface.

• Proteins Composed Predominantly of $\alpha$-Helices:

  • Myoglobin: Oxygen-binding heme protein composed almost entirely of $\alpha$-helices.

  • Lac Repressor: Contains significant $\alpha$-helical DNA-binding domains.

The $3_{10}$-Helix

• Structural Parameters:

  • A tighter, more narrow helical structure than the standard $\alpha$-helix.

  • Hydrogen Bonding Pattern: Formed between the carbonyl oxygen of amino acid $i$ ($\text{C=O}$) and the amide nitrogen hydrogen of amino acid $i+3$ ($\text{N-H}$).

  • Contains $3.0$ amino acid residues per turn, with a 10-atom ring contained within the hydrogen-bonded loop.

$\beta$-Strands and $\beta$-Sheets

• $\beta$-Strand Geometry:

  • A $\beta$-strand is an extended polypeptide chain conformation rather than a tightly coiled helix.

  • Torsion Angles: Located in the upper-left region of the Ramachandran plot, centered at $\phi = -119^\circ \text{ to } -139^\circ$ and $\psi = +113^\circ \text{ to } +135^\circ$.

  • Periodicity: The distance between adjacent amino acid residues along a fully extended $\beta$-strand is approximately $3.5\,\text{\AA}$, yielding a repeat distance of $7.0\,\text{\AA}$ for two amino acid residues.

  • Sidechains alternate sequentially above and below the plane of the mainchain strand.

• $\beta$-Sheet Architecture:

  • Formed by inter-strand hydrogen bonding between $\text{N-H}$ and $\text{C=O}$ backbone groups of two or more adjacent $\beta$-strands.

  • Antiparallel $\beta$-Sheet: Adjacent $\beta$-strands run in opposite N-to-C directions. Hydrogen bonds between strands are straight, linear, and perpendicular to the strand axis ($\text{180}^\circ$ alignment).

  • Parallel $\beta$-Sheet: Adjacent $\beta$-strands run in the same N-to-C direction. Hydrogen bonds between strands are slanted/angled.

  • Mixed $\beta$-Sheet: Contains combinations of parallel and antiparallel strand arrangements.

• Three Elements of $\beta$-Sheet Stabilization:

  1. Inter-strand backbone hydrogen bonds between adjacent $\beta$-strands.

  2. Favorable sidechain orientations and backbone torsion angles ($\phi = -119^\circ \text{ to } -139^\circ$, $\psi = +113^\circ \text{ to } +135^\circ$).

  3. Hydrophobic interactions formed by packing $\beta$-sheets against other secondary structure elements or within a $\beta$-barrel motif.

Reverse Turns and Loops

• Reverse (or $\beta$) Turns:

  • Compact structural elements that reverse the direction of a polypeptide chain by $180^\circ$ across four amino acid residues ($i$, $i+1$, $i+2$, $i+3$).

  • Stabilization: Hydrogen bond formed between the carbonyl oxygen of residue $i$ ($\text{C=O}$) and the amide nitrogen hydrogen of residue $i+3$ ($\text{N-H}$).

  • Frequently positioned on the surface of globular proteins, connecting adjacent $\alpha$-helices or $\beta$-strands.

• Loops:

  • Flexible, non-repeating surface structures that lack regular periodic hydrogen bonding patterns but connect distinct secondary structure units.

Collagen Triple Helix

• Primary Structure Sequence Characteristics:

  • Consists of a strict, repeating amino acid tripeptide sequence motif: $\text{-Gly-Pro-Hyp-}$ or $\text{-Gly-X-Y-}$.

  • Every third residue must be Glycine ($\text{Gly}$) due to extreme spatial constraints at the central junction of the triple helix.

  • Contains high proportions of Proline ($\text{Pro}$) and modified Hydroxyproline ($\text{Hyp}$).

  • Example amino acid sequence:

    • 13-Gly-Pro-Met-Gly-Pro-Ser-Gly-Pro-Arg-

    • 22-Gly-Leu-Hyp-Gly-Pro-Hyp-Gly-Ala-Hyp-

    • 31-Gly-Pro-Gln-Gly-Phe-Gln-Gly-Pro-Hyp-

    • 40-Gly-Glu-Hyp-Gly-Glu-Hyp-Gly-Ala-Ser-

    • 49-Gly-Pro-Met-Gly-Pro-Arg-Gly-Pro-Hyp-

    • 58-Gly-Pro-Hyp-Gly-Lys-Asn-Gly-Asp-Asp-

• Supramolecular Triple-Helical Architecture:

  • Individual polypeptide chains adopt an extended, left-handed polyproline-like helix conformation.

  • Three individual left-handed helical chains wrap around a common central axis to form a tight, right-handed triple superhelix (collagen triple helix).

  • Interstrand hydrogen bonds link the backbone $\text{N-H}$ groups of Glycine residues on one chain to carbonyl $\text{C=O}$ groups on neighboring chains.

  • Water-mediated hydrogen bond networks involving the hydroxyl ($\text{-OH}$) group of Hydroxyproline ($\text{Hyp}$) provide essential structural stabilization.