9-19 Protein Sequencing, Homology, and Secondary Structure

Homology

Homology Search: A process used to compare protein or DNA sequences to find regions of similarity, suggesting evolutionary relationships or shared functions.

Orthologs

Definition: Proteins from different species that have the same function.
Example: Cytochrome C (cyto c)
- Present in the mitochondria of all eukaryotes, essential for ATP production, acting as a carrier between Complex III and Complex IV in the electron transport chain.
- Comparison of human cytochrome c sequence with other organisms reveals amino acid variations:
  - Snake: $14$ variations.
  - Fish (e.g., Catfish): $18$ variations.
  - Snail: $29$ variations.
  - Insect: $31$ variations.
  - Yeast (lower organism, single-cell eukaryote): Greater than $41$ amino acid variations.
- These variations demonstrate evolutionary divergence, with lower organisms showing more differences from humans.
Phylogenetic Tree: A diagram representing evolutionary relationships between organisms based on sequence variations of a homologous protein (like cytochrome c).
- Shows branching patterns, with organisms like chimps and humans being very close at the tips, indicating minimal variation in cytochrome c sequences (often $0$ variations).
- Suggests a common immediate ancestor.
- Human and chimp DNA sequences are approximately $99\%$ identical, reinforcing close evolutionary relatedness.

Paralogs

Definition: Proteins from the same species or organism that have the same function.
Example: Serine Proteases
- A group of enzymes responsible for protein degradation.
- Includes:
  - Trypsin (dietary protease in the stomach).
  - Chymotrypsin (dietary protease in the stomach).
  - Elastase (dietary protease in the stomach).
  - Thrombin (present in the blood, involved in clotting).
- These are different proteins within the same species (human) but perform the same general function of proteolysis.
Example: Ancestor Globin Gene Divergence
- An ancestral globin gene diverged into different globin proteins within an organism.
- Myoglobin: Not much change from the ancestral globin, typically composed of a single subunit with $153$ amino acids.
- Hemoglobin: Diverged into alpha ( $\alpha$ ) and beta ( $\beta$ ) subunits.
  - Alpha ( $\alpha$ ) and Beta ( $\beta$ ) globin have about $68\%$ sequence identity (identical amino acids), with the rest being variable.
  - Alpha ( $\alpha$ ) globin and Myoglobin have about $8\%$ sequence identity (identical amino acids).
- This indicates that alpha globin is evolutionarily more distant from myoglobin than beta globin is, suggesting beta globin is closer to myoglobin in terms of sequence.

Exceptions to Orthologs

Identity does not always mean identical function.
Example: Hen Lysozyme and Human Lactalbumin
- Hen Lysozyme: Has $129$ amino acids. Its function is to cut sugar bonds in bacterial cell walls (antibacterial).
- Human Lactalbumin: Has $123$ amino acids. Its function is to regulate the synthesis of lactose in mammary glands.
- They share $48\%$ amino acid sequence identity, indicating some conserved regions (e.g., related to oxygen binding, common structural folds).
- However, despite significant sequence identity, their specific functions are entirely different.

Exceptions to Paralogs (Mutant Proteins)

**A single mutation can drastically alter protein function within the same species.
Example 1: Mutant RAS (RASm)
- Normal RAS: A protein involved in cell signaling.
- Mutant RAS: A mutation at the glycine $12$ position, converting it to valine ( $\text{Gly}12\rightarrow \text{Val}12$ ).
- This mutant RAS (oncogenic) binds to GTP irreversibly, leading to uncontrolled cell proliferation and tumor formation.
- It's the same protein (RAS) but a single amino acid change leads to a profoundly different and pathological function.
Example 2: Sickle Cell Anemia
- Normal Hemoglobin (HbA): The beta ( $\beta$ ) subunit of hemoglobin has a glutamic acid ( $\text{E}$ ) at position $6$ ($\beta- \text{Glu6} $) (or valine, depending on the reference in the transcript, but the correction states E6).<ul><li>Binds oxygen efficiently.</li><li>Present in normal red blood cells (RBCs).</li></ul></li><li>Sickle Cell Hemoglobin (HbS): A mutation at position$ 6 $of the beta ($ \beta $) subunit, where glutamic acid ($ \text{E} $) is changed to valine ($ \text{V} $) (i.e.,$ \beta-\text{Glu}6\rightarrow \beta-\text{Val}6 $).<ul><li>This mutation causes the hemoglobin to form a crescent-like (sickle) shape instead of the normal biconcave disc.</li><li>Consequences:<ul><li>Cannot bind to oxygen efficiently.</li><li>Sickled RBCs cluster together in arteries.</li><li>Macrophages degrade these abnormal cells, leading to anemic conditions.</li></ul></li><li>This condition is called Sickle Cell Anemia, first identified in$ 1929 $in Chicago.</li></ul></li></ul></li></ul><h5 id="d8a2fc97-5c2e-4209-8969-ad12c49524be" data-toc-id="d8a2fc97-5c2e-4209-8969-ad12c49524be" collapsed="false" seolevelmigrated="true">Protein Structure: Secondary Structure</h5><ul><li>Nucleation Sites: Regions within a protein sequence (e.g.,$ 2-4 $hydrophobic amino acids) that initiate protein folding by burying themselves in the interior of the protein to avoid water.</li><li>Secondary Structure: Localized, recurring structures formed by regular hydrogen bonding patterns between amino acids in the polypeptide backbone.<ul><li>These structures form spontaneously without external energy input.</li></ul></li><li>Types of Secondary Structures:<ul><li>Alpha Helices ($ \alpha $helices): Right-handed ($ \alpha \text{R} $) and left-handed ($ \alpha \text{L} $) helical structures. The common form is$ \alpha \text{R} $. A less common variant is the Pi helix ($ \pi \text{helix} $).</li><li>Beta Bends (Beta Turns): Short, U-shaped regions connecting two strands of a protein, allowing the polypeptide chain to reverse direction. Classified as Type I and Type II.</li></ul></li></ul><h5 id="d4a701cb-553a-42cd-9852-1828c3423be1" data-toc-id="d4a701cb-553a-42cd-9852-1828c3423be1" collapsed="false" seolevelmigrated="true">Factors Affecting Protein Folding Patterns</h5><ul><li>Peptide Bond Characteristics:<ul><li>The peptide bond (between the carbonyl carbon and the amide nitrogen) has about a$ 40\% $double bond character due to the delocalization of the lone pair of electrons from the nitrogen atom. This can be calculated from bond length comparison (e.g., with single bond length).</li><li>This partial double bond character means the peptide bond is rigid and planar, preventing rotation around the C-N bond.</li><li>The bonds that can rotate are the$ \text{C}\alpha-\text{N} $bond (phi,$ \phi $angle) and the$ \text{C}\alpha-\text{C} $bond (psi,$ \psi $angle).</li><li>Rotation is restricted to specific angles (e.g.,$ \pm 90^\circ $to$ \pm 45^\circ $) to avoid steric clashes, making only certain$ \phi $and$ \psi $angle combinations favorable.</li></ul></li><li>Ramachandran Plot:<ul><li>A theoretical plot (later confirmed by NMR studies) of$ \phi $versus$ \psi $angles for amino acid residues in protein structures.</li><li>Plots show regions representing favorable and unfavorable conformations, defining the allowable angles for secondary structures.</li><li>Key regions correspond to alpha helices ($ \alpha \text{helices} $), beta sheets, and beta turns ($ \beta \text{turns} $).</li></ul></li></ul><h5 id="5fec47a1-fa26-4973-878a-568e1daf2d78" data-toc-id="5fec47a1-fa26-4973-878a-568e1daf2d78" collapsed="false" seolevelmigrated="true">Alpha Helix Details</h5><ul><li>Types of Alpha Helices: Based on the number of residues per turn and the number of atoms in the hydrogen-bonded ring.<ul><li>$ 3.6_{13} $helix (standard$ \alpha \text{helix} $)</li><li>$ 3.0_{10} $helix</li><li>$ 4.4_{16} $helix (also called$ \pi \text{helix} $)</li></ul></li><li>$ 3.6_{13} $Alpha Helix (Standard Alpha Helix):<ul><li>Average of$ 3.6 $amino acid residues per helical turn.</li><li>Involves a$ 13 $-atom ring formed by hydrogen bonding.</li><li>Hydrogen Bonding Pattern: The carbonyl oxygen ($ \text{C}=\text{O} $) of residue$ n $forms a hydrogen bond with the amide hydrogen ($ \text{N}-\text{H} $) of residue$ n+4 $(i.e., every fourth$ C\alpha $involves a hydrogen bond).</li><li>Location: Often found at the beginning of an alpha helix.</li><li>Amino Acid Distance: The distance between consecutive amino acids along the helix axis (pitch per residue) is approximately$ 0.15 \text{ nm} $or$ 1.5 \text{ Å} $.<ul><li>Pitch of one helical turn (for$ 3.6 $residues) is$ 3.6 \times 0.15 \text{ nm} \approx 0.54 \text{ nm} $(or$ 5.4 \text{ Å} $).</li></ul></li></ul></li><li>$ 3.0_{10} $Helix:<ul><li>$ 3.0 $amino acid residues per turn.</li><li>$ 10 $-atom ring in the hydrogen bond.</li><li>Often found in the interior part of a protein.</li></ul></li><li>$ 4.4_{16} $Helix (Pi Helix):<ul><li>$ 4.4 $amino acid residues per turn.</li><li>$ 16 $-atom ring in the hydrogen bond.</li><li>A broader helix (e.g., found toward the C-terminal).</li></ul></li><li>Formula for Hydrogen Bonds in a Helix: For a helix with$ n $amino acid residues, the number of hydrogen bonds is given by$ n-4 $(assuming standard$ i\to i+4 $bonding).</li></ul><h5 id="8552bf87-cbdf-49a4-aa38-8e864175441b" data-toc-id="8552bf87-cbdf-49a4-aa38-8e864175441b" collapsed="false" seolevelmigrated="true">Beta Strands and Beta Sheets</h5><ul><li>Beta strands can be arranged in parallel ($ \text{N}\to\text{C} $and$ \text{N}\to\text{C} $) or antiparallel ($ \text{N}\to\text{C} $and$ \text{C}\to\text{N}$$) configurations within beta sheets. These sheets are connected by loops or turns (like beta bends).