8/31 Lecture

Course Expectations and Primary Structure Sequence Analysis

• Assessment Expectations for Amino Acids:

  • Functional groups present on each amino acid must be thoroughly understood.

  • Classifications of each amino acid must be known.

  • Side-chain chemical properties and their impact on protein structure and function must be mastered.

• Primary Structure Overview and Genome Sequencing:

  • Primary structure represents the linear amino acid sequence of a polypeptide chain.

  • Modern genome sequencing technology enables rapid sequencing of entire genomes.

  • Computer algorithms scan sequenced genomes for start genes and termination points, translating gene sequences directly into primary protein sequences.

• Sequence Alignment and Myosin Gene Evolution:

  • Alignment tools allow comparison of primary sequences across different genes within the same species or across different species.

  • Myosin Gene Family Example:

  • The human genome contains approximately $15$ distinct myosin genes.

  • Specific myosin genes exist for distinct tissue types and functional roles: skeletal muscle, cardiac muscle, smooth muscle, and cell motility/crawling.

  • Paralogs: Multiple myosin genes within the same organism (e.g., human myosin genes compared to one another) are paralogs. They share core conserved functions—binding actin, driving contraction, and moving via ATP hydrolysis—but possess subtle amino acid variations that confer distinct functional properties.

  • Orthologs: Corresponding myosin genes compared across different species (e.g., comparing human myosin to dog or horse myosin) are orthologs.

Evolutionary Analysis and Secondary Structure Principles

• Phylogenetic Trees from Protein Alignments:

  • Aligning primary sequences of a conserved protein across $30\text{ to }40$ different organisms allows computational generation of phylogenetic trees.

  • Cytochrome c Example: Sequence alignments of cytochrome c across diverse species illustrate evolutionary relationships.

  • Branch Length Significance: The lengths of branches on a phylogenetic tree correspond directly to evolutionary distance and time.

  • Multi-Protein Evolutionary Analysis: To derive overarching conclusions regarding organismal evolution, researchers analyze multiple distinct proteins to confirm that independent trees yield consistent evolutionary relationships.

• Fundamental Definition of Secondary Structure:

  • Secondary structure imparts three-dimensional spatial dimensionality to polypeptide chains.

  • It requires regular, repeating dihedral angles of rotation along the backbone maintained over a sufficient consecutive stretch of amino acids.

  • The two dominant secondary structure elements are the alpha helix and the beta sheet (composed of individual beta strands).

  • Protein composition varies: some proteins are purely alpha-helical, others consist entirely of beta sheets, and most feature combinations of helices, strands, and sheets.

• Connectors and Random Coils:

  • Not every amino acid in a folded protein is part of a strict secondary structure element.

  • Linker or connector regions connect separate secondary structure elements (e.g., helix-to-helix or strand-to-strand).

  • These connecting regions are termed random coils.

  • Random coils lack regular, repeating secondary structure angles, yet they are essential for proper protein folding.

  • Random coils exhibit structural flexibility ("floppiness") in three-dimensional space because their backbone atoms are not constrained to fixed positions.

Backbone Geometry, Peptide Bonds, and Ramachandran Plots

• Polypeptide Backbone (Main Chain) Definitions:

  • The main chain or polypeptide backbone includes all backbone atoms excluding the amino acid side chains (main chain and polypeptide backbone are used interchangeably).

  • Along the backbone, the covalent bond order repeats sequentially: alpha carbon ($\text{C}_\alpha$) to carbonyl carbon ($\text{C}$), carbonyl carbon ($\text{C}$) to amide nitrogen ($\text{N}$), and amide nitrogen ($\text{N}$) to alpha carbon ($\text{C}_\alpha$).

• Peptide Bond Planarity and Resonance:

  • The peptide bond links the carbonyl carbon to the amide nitrogen ($\text{C-N}$).

  • Resonance occurs across the four atoms of the peptide unit (carbonyl oxygen, carbonyl carbon, amide nitrogen, and amide hydrogen).

  • Resonance imparts partial double-bond character to the peptide bond.

  • Consequently, there is no rotation around the peptide bond itself; the four atoms of the peptide bond reside in a rigid planar configuration.

• Dihedral Angles (Phi and Psi):

  • Rotation in the polypeptide backbone occurs exclusively around the two single bonds adjacent to the alpha carbon ($\text{C}_\alpha$):

  • Phi ($\phi$): The angle of rotation around the bond between the amide nitrogen and the alpha carbon ($\text{N-C}_\alpha$).

  • Psi ($\psi$): The angle of rotation around the bond between the alpha carbon and the carbonyl carbon ($\text{C}_\alpha\text{-C}$).

  • Dihedral angles are defined on a system ranging from $0^\circ$ to $+180^\circ$ and $0^\circ$ to $-180^\circ$ (a $\pm 180^\circ$ system).

• Ramachandran Plots and Steric Hindrance:

  • Theoretical free rotation around $\phi$ and $\psi$ is severely restricted due to steric collisions between backbone atoms and side chains.

  • The Ramachandran plot maps all theoretical combinations of $\phi$ and $\psi$ angles to identify permitted vs. sterically forbidden regions:

  • Yellow Regions: Represent sterically forbidden $\phi$/$\psi$ combinations caused by atomic collisions ($70\%$ or more of all possible $\phi$/$\psi$ angle combinations are excluded).

  • Light Blue Regions: Represent disfavored or strained conformations where minor atomic crowding occurs.

  • Dark Blue Regions: Represent optimal, fully allowed $\phi$/$\psi$ conformations free of steric collisions.

  • The severe restriction of allowed $\phi$/$\psi$ angles explains why protein secondary structures are predominantly restricted to alpha helices and beta sheets.

  • Left-handed alpha helices appear in an allowed region on the Ramachandran plot, but left-handed alpha helices essentially do not occur in natural proteins.

Structural Features of the Right-Handed Alpha Helix

• Helical Parameters and Handedness:

  • Naturally occurring alpha helices are strictly right-handed.

  • Pitch and turn density: An alpha helix contains approximately $3.5$ (or ~$3.6$) amino acids per complete turn.

  • Linear spacing: A full turn spans a vertical distance of $5.4\,\text{\AA}$ along the helical axis.

  • Central core: The central axis inside the helix cylinder forms a narrow physical opening of $4\text{ to }5\,\text{\AA}$; it is completely inert and inaccessible to diffusing molecules.

• Backbone Hydrogen Bonding Network:

  • The alpha helix is stabilized predominantly by hydrogen bonds along the polypeptide main chain.

  • Hydrogen bonds form between the carbonyl oxygen of residue $n$ and the amide hydrogen of residue $n+4$ ($n \rightarrow n+4$ rule).

  • These hydrogen bonds align parallel to (in line with) the central longitudinal axis of the helix.

  • Interior amino acids in an alpha helix participate in two hydrogen bonds: one extending upward and one extending downward along the helix axis.

• Side Chain Orientation and Heptad Repeats:

  • Side chains project outward and downward from the helical backbone cylinder.

  • Heptad Repeat Pattern: The alpha helix exhibits a repeating structural periodicity every $7$ residues, designated by positions $a, b, c, d, e, f, g$ (or numbers $1$ to $7$).

  • Residue $1$ aligns vertically above residue $8$, and residue $2$ aligns vertically above residue $9$, forming a vertical seam along the helix length.

  • Residues at positions $1$ and $8$ are too far apart for direct side-chain interaction, but side chains at positions $1$ and $4$ can interact directly.

  • Side Chain Stabilization: Oppositely charged side chains at positions $1$ and $4$ (e.g., a positively charged side chain at position $1$ and a negatively charged side chain at position $4$) form stabilizing ionic bonds (salt bridges).

  • In coiled-coil higher-order structures, residues at hydrophobic positions $a$ and $d$ stack along the internal seam to mediate helix-helix packing.

Amino Acid Propensities, Helix Breakers, and Helix Capping

• Amino Acid Residue Preferences:

  • Small hydrophobic side chains, such as Alanine and Leucine, exhibit high conformational propensity for alpha helix formation.

• Helix Breakers:

  • Proline: Acts as a severe helix breaker because its cyclic three-carbon side chain covalently links back to its backbone nitrogen, forming a rigid ring structure. This rigid geometry prevents the backbone from adopting the required $\phi$ angle for an alpha helix.

  • Glycine: Acts as a helix breaker because its minimal side chain (a single hydrogen atom) confers excessive conformational flexibility, thermodynamically destabilizing the constrained helical structure.

• Helix Capping:

  • Amino acids at the extreme N-terminal and C-terminal ends of an alpha helix lack upstream or downstream backbone hydrogen bonding partners within the helix.

  • Helix capping refers to specific amino acid interactions at these terminal positions that provide alternative hydrogen bonding and stabilizing interactions to seal the helix ends.

Structural Characteristics of Beta Strands and Beta Sheets

• Beta Strand Backbone Geometry:

  • Individual beta strands exhibit a pleated or zigzag appearance caused by the tetrahedral geometry of the alpha carbons ($\text{C}_\alpha$) combined with the planar geometry of peptide bonds.

  • Polypeptide directionality is designated by an arrow pointing from the N-terminus to the C-terminus.

  • Amino acids with larger side chains display a conformational preference for beta strands and beta sheets.

• Side Chain and Hydrogen Bond Orientation in Beta Sheets:

  • Beta sheets are composed of multiple individual beta strands aligned side by side.

  • Side chains project perpendicularly above and below the plane of the sheet in an alternating pattern (up, down, up, down).

  • Hydrogen bonds extend laterally (sideways) between backbone amide nitrogens and carbonyl oxygens of adjacent strands.

Parallel vs. Antiparallel Beta Sheets

• Parallel Beta Sheets:

  • Adjacent beta strands run in the same N-to-C directional orientation.

  • Hydrogen bonds between parallel strands exhibit a bent (non-linear) geometry.

  • Non-linear hydrogen bonds in parallel sheets are inherently weaker than linear hydrogen bonds.

  • Connecting parallel strands requires long loop or linker regions in the primary sequence (e.g., $20\text{ to }25$ amino acids, alpha helices, or complex coils) to loop around and enter the next strand in the same direction.

• Antiparallel Beta Sheets:

  • Adjacent beta strands run in opposite directional orientations (N-to-C adjacent to C-to-N).

  • Hydrogen bonds between antiparallel strands exhibit a linear ($180^\circ$) geometry.

  • Linear hydrogen bonds confer greater structural stability compared to parallel sheets.

  • Antiparallel strands can be connected by short primary sequence linkers consisting of just a few amino acids.

Beta Turns and Proline Cis/Trans Isomerism

• Beta Turn Definition and Function:

  • A beta turn is a tight secondary structure motif that enables the polypeptide chain to make a sharp $180^\circ$ turn in direction.

  • Beta turns frequently connect adjacent strands in antiparallel beta sheets.

  • A beta turn spans exactly $4$ amino acid residues and is stabilized by a hydrogen bond between the carbonyl oxygen of residue $1$ and the amide hydrogen of residue $4$.

• Types of Beta Turns:

  • Type I Beta Turn: Features Proline at position $2$ and Glycine at position 3$.\n * **Type II Beta Turn:** Features **Glycine** at position 3.\n * Glycine is critical at position 3$because its small side chain (hydrogen) eliminates steric hindrance, accommodating the tight backbone rotation required for the$180^\circ bend.\n\n* **Proline Cis/Trans Isomerization:**\n * Partial double-bond character restricts peptide bond rotation, giving rise to *cis* and *trans* isomers.\n * The vast majority of non-proline peptide bonds exist exclusively in the *trans* configuration.\n * Proline's cyclic side chain reduces the energy difference between *cis* and *trans* forms.\n * While most proline residues remain *trans*, Type I beta turns specifically utilize Proline in the **cis configuration** to facilitate the tight directional turn.\n\n# Tertiary Structure and Side Chain Interactions\n\n* **Definition of Tertiary Structure:**\n * Tertiary structure represents the complete three-dimensional folded conformation of a single polypeptide chain.\n * It is driven primarily by interactions between amino acid side chains.\n * Tertiary folding brings residues that are far apart in the primary sequence (e.g., residue 5$and residue$50$$) into close spatial proximity.

• Types of Side Chain Interactions:

  • Hydrophobic Effect and Van der Waals Forces: Nonpolar side chains aggregate together in the interior to avoid water.

  • Hydrogen Bonding: Polar side chains interact with other polar side chains.

  • Disulfide Bonds: Cysteine residues form covalent disulfide bonds under oxidizing conditions.

  • Ionic Bonds / Salt Bridges: Oppositely charged side chains form electrostatic interactions.

  • Side Chain to Backbone Interactions: Polar side chains form hydrogen bonds with backbone amide nitrogens or carbonyl oxygens.

Protein Classification and Fibrous Proteins: Keratin

• Four Structural Classes of Proteins:

  1. Fibrous Proteins: Long, structural proteins dominated by hydrophobic residues; water-insoluble; form large macromolecular assemblies.

  2. Globular Proteins: Spherical, compact functional proteins; highly soluble in aqueous solutions.

  3. Membrane Proteins: Proteins embedded within or spanning lipid bilayers.

  4. Intrinsically Disordered Proteins: Proteins lacking a defined, stable three-dimensional tertiary structure.

• Alpha-Keratin Structure and Assembly:

  • An individual keratin monomer is a long, right-handed alpha helix.

  • Two keratin right-handed helices wrap around each other to form a dimer called a coiled-coil.

  • Crucially, the two right-handed helices intertwine in a left-handed orientation to form the coiled-coil.

  • Coiled-coils assemble side-by-side in a staggered arrangement into large supramolecular structures.

  • Assembly is stabilized by extensive covalent disulfide cross-links between cysteine residues in extracellular oxidizing environments.

• Biochemical Process of Hair Perming / Curling:

  • Straight hair contains abundant native disulfide cross-links holding keratin fibers in place.

  • Step 1 (Reduction): Application of a reducing agent breaks existing covalent disulfide bonds (accompanied by a strong, distinct odor).

  • Step 2 (Heat Application): Hair is wrapped around curlers, and heat is applied to disrupt weak non-covalent interactions (hydrogen bonds).

  • Step 3 (Cooling): The temperature is lowered, allowing non-covalent interactions to re-form in the newly curled shape.

  • Step 4 (Oxidation): An oxidizing agent is added to re-form covalent disulfide bonds in new positions, permanently locking hair in the curled configuration.

Fibrous Proteins: Collagen Structure and Cross-Linking

• Collagen Amino Acid Composition:

  • Collagen is a major structural protein with a specialized primary sequence rich in Proline, Hydroxyproline (proline modified by hydroxyl group addition), and Glycine.

  • Repeating sequence motifs form an extended, unique left-handed helix.

  • Individual collagen left-handed helical chains are historically referred to as "alpha chains" (note: despite the name, an alpha chain is a left-handed helix, NOT a right-handed alpha helix).

• Collagen Triple Helix Rope Structure:

  • Three left-handed alpha chains wrap around one another to form a superhelical collagen triple helix.

  • While individual strands are left-handed, the three strands wrap around each other in a right-handed orientation (resembling a braided rope).

• Higher-Order Stacking and Covalent Cross-Linking:

  • Collagen triple helices stack and aggregate into larger collagen fibrils and bundles.

  • Unlike keratin (which uses disulfide bonds), collagen assemblies are cross-linked covalently via Lysine and Hydroxylated Lysine (Hydroxylysine) residues.

Principles of Globular Protein Folding

• Characteristics of Globular Proteins:

  • Globular proteins represent the largest class of proteins.

  • They adopt compact, roughly spherical three-dimensional structures.

  • Globular proteins contain diverse structural mixtures of alpha helices, beta strands, beta sheets, turns, and random coils.

• Thermodynamic Driving Forces of Globular Folding:

  • Operating in aqueous cellular environments, folding is driven by the hydrophobic effect: nonpolar amino acid side chains are sequestered into the dense internal core away from water.

  • Folding maximizes favorable internal weak chemical interactions, including interior hydrogen bonds and ionic salt bridges.

  • Maximizing internal non-covalent interactions minimizes free energy and yields maximum thermodynamic stability.