Proteins

Proteins: Synthesis and Folding

Part 1: Synthesis and Folding

From Gene to Protein

• Eukaryotic protein synthesis stages:

  • Cytoplasm: The site where translation occurs and where mature mRNA is translated into polypeptide chains by ribosomes.

  • Nucleus: The command center containing the genome; it is the site for transcription and initial RNA processing.

  • Introns: Non-coding intervening sequences within a gene that are transcribed but subsequently removed by splicing.

  • Exons: The coding sequences that remain after splicing and are expressed to determine the amino acid sequence.

  • DNA: The double-stranded template containing the genetic code ($A, T, C, G$).

  • Transcription unit: A stretch of DNA that is transcribed into an RNA molecule; it begins at a promoter and ends at a terminator.

  • Primary RNA transcript (pre-mRNA): The direct product of transcription, containing both introns and exons, before any modifications.

Transcription Process

1. Transcription: Catalyzed by RNA Polymerase II, this process uses one strand of DNA as a template to synthesize a complementary RNA strand.

2. 5' Capping: The addition of a $7$-methylguanosine cap to the $5'$ end of the pre-mRNA; this prevents degradation and serves as a "binding site" for ribosomes.

3. RNA Splicing: Orchestrated by the spliceosome (a complex of snRNPs), this removes introns and ligates exons together.

4. 3' Polyadenylation: An enzyme adds a sequence of $100-250$ adenine nucleotides (Poly-A tail) to the $3'$ end, facilitating nuclear export and mRNA stability.

5. mRNA Recognition: The combination of the $5'$ cap and the Poly-A tail signals that the mRNA is intact and ready for translation.

6. Export of mRNA: Mature mRNA exits the nucleus through Nuclear Pore Complexes (NPCs) into the cytoplasm.

Translation Process

• Involves decoding the genetic code (codons) into a specific sequence of amino acids:

  • Ribosomes: Composed of a small ($40S$) and large ($60S$) subunit in eukaryotes. They scan mRNA for the start codon ($AUG$).

  • tRNA and Aminoacyl-tRNA Synthetases: tRNA molecules carry specific amino acids to the ribosome; synthetases ensure the correct amino acid is attached to the correct tRNA.

  • A, P, and E Sites: The ribosome contains three sites for tRNA binding: the A (Aminoacyl) site, the P (Peptidyl) site, and the E (Exit) site.

Protein Folding Initiation

• Co-translational folding: Folding often begins as the N-terminus emerges from the ribosome exit tunnel.

  • Polypeptide chain: The linear polymer of amino acids linked by covalent peptide bonds ($CO-NH$).

  • N-terminal domain: The first part of the protein synthesized ($5'$ end of mRNA); targets for early folding or signal sequence recognition.

  • C-terminal domain: The final portion of the protein synthesized ($3'$ end of mRNA).

• Post-translational folding: Final globular or fibrous structures are achieved only after the full chain is released.

Protein Structure

Amino Acid Structure

• General Formula: Each amino acid has a central alpha-carbon ($C_\alpha$) bonded to:

  • An amino group ($-NH_2$).

  • A carboxyl group ($-COOH$).

  • A hydrogen atom.

  • A variable side chain (R group) that defines chemical properties.

Types of Amino Acids

• Nonpolar (Hydrophobic): Tend to be buried in the protein interior (e.g., Alanine $Ala$, Valine $Val$, Methionine $Met$, Tryptophan $Trp$).

• Polar (Uncharged): Form hydrogen bonds with water (e.g., Serine $Ser$, Threonine $Thr$, Glutamine $Gln$, Tyrosine $Ty r$).

• Charged (Hydrophilic): Located on the protein surface to form ionic bonds/salt bridges (e.g., Aspartic acid $Asp, -$ charge, Lysine $Lys, +$ charge).

Levels of Protein Structure

1. Primary structure: The linear sequence of amino acids.

2. Secondary structure: Folding into $\alpha$-helices and $\beta$-sheets, stabilized by hydrogen bonds between the backbone carbonyl and amide groups.

3. Tertiary structure: The full 3D conformation stabilized by hydrophobic interactions, van der Waals forces, ionic bonds, and disulfide bridges between Cysteine residues.

4. Quaternary structure: The spatial arrangement of multiple polypeptide subunits (e.g., Hemoglobin).

Chaperones and Protein Folding

Molecular Chaperones

• Function: These are "folding helpers" that prevent inappropriate interactions; they do not carry the information for folding but facilitate the process.

• HSP 70: Acts early; binds to exposed hydrophobic patches on newly synthesized proteins using ATP to prevent aggregation.

• HSP 60 (Chaperonins): Form a massive "isolation chamber" (like the GroEL/ES system in bacteria) where a single misfolded protein can fold in a protected environment.

Enzymes in Folding

• Protein Disulfide Isomerase (PDI): Catalyzes the formation and shuffling of disulfide bonds in the Endoplasmic Reticulum.

• Peptidyl Prolyl Isomerase (PPI): Catalyzes the rotation about proline peptide bonds, often a rate-limiting step in folding.

Part 2: Modification and Degradation

Post-Translational Modifications (PTMs)

• Proteolytic cleavage: The trimming of the polypeptide chain to activate a protein (e.g., Zymogens like Pepsinogen or hormones like proinsulin).

• Glycosylation:

  • N-linked: Attachment of oligosaccharides to Asparagine in the ER.

  • O-linked: Attachment to Serine or Threonine, primarily in the Golgi apparatus.

• Lipid Anchors: Covalent attachment of lipids (e.g., GPI anchors) to target proteins to the cell membrane.

• Phosphorylation: The addition of a phosphate group ($PO_4^{3-}$) by Kinases to change protein activity; reversed by Phosphatases.

Ubiquitin-Proteasome Pathway

1. Ubiquitination Cascade:

   • E1 (Activating Enzyme): Uses ATP to activate Ubiquitin.

   • E2 (Conjugating Enzyme): Carries the activated Ubiquitin.

   • E3 (Ligase): The specificity factor that recognizes the target "degron" sequence and transfers Ubiquitin to the substrate.

2. Polyubiquitination: A chain of at least four Ubiquitins linked via Lysine 48 (K48) targets the protein for the proteasome.

3. The 26S Proteasome: A massive complex with a 19S regulatory cap (recognizes and unfolds the protein) and a 20S catalytic core (breaks the protein into short peptides).

Quality Control and Disease

• ER-Associated Degradation (ERAD): Misfolded proteins in the ER are retro-translocated back to the cytoplasm for proteasomal degradation.

• Cystic Fibrosis: Often caused by a mutation ($DF508$) in the CFTR protein; the protein is slightly misfolded and is destroyed by quality control even though it might still be functional.