Protein Structure, Function, and Regulation Comprehensive Lecture Two Notes: Hierarchical Organization, Enzymes, and Regulation

Hierarchical Structure of Proteins and Supramolecular Assemblies

Levels of Protein Organization:
- Primary Structure: The specific amino acid sequence of the protein, listed from the N-terminus to the C-terminus.
- Secondary Structure: Localized folding within a single polypeptide chain, characterized by stable spatial arrangements of segments. It is maintained by hydrogen bonds between amide and carbonyl groups.
- Tertiary Structure: The overall three-dimensional conformation of a single polypeptide chain. It is stabilized by side-chain interactions including hydrophobic interactions, ionic bonds, hydrogen bonds, and covalent disulfide bridges.
- Quaternary Structure: The overall structure of a protein complex consisting of more than one polypeptide chain (multimeric proteins).
- Supramolecular Structure: Large-scale assemblies of proteins with themselves or other macromolecules (e.g., RNA in Ribonuclear Protein or RNP particles).
Substantive Protein Functions:
- Regulation: Serving as on/off switches for cellular processes.
- Structure: Providing internal cellular frameworks.
- Movement: Facilitating movement of the cell itself or transport within the cell (e.g., along microtubules).
- Catalysis: Accelerating chemical reactions via enzymes.
- Transport: Moving molecules across membranes.
- Signaling: Allowing cells to interact with the environment and other cells.

Building Blocks: Amino Acids

Isomerism and Enantiomers:
- Most amino acids exist as optical isomers or enantiomers (mirror images) due to an asymmetrical central alpha carbon ( $C_{\alpha}$ ) bound to four distinct groups.
- The Four Functional Groups:
  1. Carboxyl group ( $COO^-$ ).
  2. Amino group ( $NH_3^+$ ).
  3. Hydrogen atom ( $H$ ).
  4. Variable Side Chain ( $R$ -group).
- D and L Isomers: Named D (dextro) and L (levo) forms. While both exist in nature, only L isomers are used to synthesize proteins.
- Glysine Exception: Glycine is the only amino acid without D and L forms because it lacks four distinct functional groups (it has two hydrogens).
Amino Acid Classifications Based on R-Groups:
- Hydrophobic Amino Acids: Possess nonpolar covalent bonds (C-C or C-H) in their side chains. Includes: Alanine ( $A$ ), Valine ( $V$ ), Isoleucine ( $I$ ), Leucine ( $L$ ), Methionine ( $M$ ), Phenylalanine ( $F$ ), Tyrosine ( $Y$ ), Tryptophan ( $W$ ). Phenylalanine and Tryptophan contain hydrophobic ring structures.
- Hydrophilic Amino Acids: Side chains interact with water due to polar covalent bonds ( $O-H$ or $N-H$ ).
  - Basic (Positively Charged): Lysine ( $K$ ), Arginine ( $R$ ), Histidine ( $H$ ). Side chains carry full positive charges.
  - Acidic (Negatively Charged): Aspartate ( $D$ ) and Glutamate ( $E$ ). Side chains carry carboxyl groups with negative charges.
  - Polar (Uncharged): Serine ( $S$ ), Threonine ( $T$ ), Asparagine ( $N$ ), and Glutamine ( $Q$ ). These have polar bonds ( $O-H$ or $N-H$ ) but no full charge.
- Special Amino Acids:
  1. Cysteine ( $C$ ): Contains an $SH$ (sulfhydryl) group; can form covalent disulfide bridges ( $S-S$ ) with other cysteines.
  2. Glycine ( $G$ ): The smallest amino acid ( $R = H$ ); allows tight turns in protein structure.
  3. Proline ( $P$ ): The only amino acid where the side chain forms a ring structure by coupling to the amino group. It disrupts standard secondary structures.
Polypeptide Formation:
- Two amino acids couple via a condensation or dehydration reaction, releasing one molecule of water ( $H_2O$ ).
- A peptide bond forms between the carbon of the carboxyl group of the first amino acid and the nitrogen of the amino group of the second.
- Directionality: By convention, sequences are written from the free amino end (N-terminus) on the left to the free carboxyl end (C-terminus) on the right.

Secondary and Tertiary Structural Features

Primary Secondary Structures:
- Alpha Helix ( $\alpha$ -helix): A right-handed helical structure with $3.6$ amino acid residues per turn. Held by hydrogen bonds between the central carbon/amino groups and carboxyl oxygen.
- Beta Sheet ( $\beta$ -sheet): Consists of strands running side-by-side joined by hydrogen bonds. Strands can be Parallel (same N-to-C direction) or Anti-parallel (opposite directions).
- Beta Turn ( $\beta$ -turn): Involves $4$ amino acids to reverse the direction of the polypeptide; stabilized by hydrogen bonds.
Structural Motifs: Specific sequences of amino acids recurring across different proteins.
- Coiled-coil motif: Forms rope-like fibrous structures; important for the cytoskeleton and protein-protein interactions.
- EF hand motif: A loop-helix motif; coordinates calcium ions ( $Ca^{2+}$ ). Found in proteins like calmodulin.
- Zinc finger motif: An $\alpha$ -helix and two $\beta$ -strands coordinated around a single Zinc ion ( $Zn^{2+}$ ) using two cysteines and two histidines. Common in transcription factors.
Domains: Independent modular units of protein function/structure.
- Functional domains: Defined by a specific activity (e.g., DNA binding).
- Structural domains: Defined by a stable, distinct structural feature.
- Topological domains: Defined by their spatial relationship to the rest of the protein.
- EGF Domain Example: The Epidermal Growth Factor (EGF) domain is found in hormone precursors and unrelated proteins like Neu and tPA (tissue plasminogen activator).

Examples of Supramolecular Complexes

Replisome: Involved in nuclear DNA replication; includes DNA polymerases, primase, and helicase.
Transcription Initiation Complex: Includes RNA polymerase, General Transcription Factors (GTFs), and the large Mediator complex.
Spliceosome: An RNP complex for mRNA splicing; involves Small Nuclear RNAs (snRNAs) and proteins acting on pre-mRNA.
Nucleoporin Complex: Comprised of about $100$ distinct proteins forming pores through the nuclear envelope to regulate import/export.
Ribosome: The best-studied RNP; consists of a large and small subunit made of rRNA and ribosomal proteins.
Proteasome: A machine for regulated protein degradation in the cytoplasm.
Sarcomere: The contractile unit involving thick (myosin) and thin (actin) filaments.

Protein Folding and Chaperones

Self-Refolding: Small, single-polypeptide proteins like RNase A (Ribonuclease A) are remarkably stable and can renature autonomously after heat denaturation.
Molecular Chaperones: Proteins that use ATP hydrolysis to promote proper folding of newly synthesized proteins in vivo.
- Hsp70 (Heat Shock Protein 70): Binds to unfolded polypeptides. Its cycle involves DNA J, Hsp40, GRPE, and BAG1. ATP hydrolysis causes a conformational change in the substrate binding domain from an open to a closed state to induce folding.
- Chaperonins (e.g., GroEL/GroES in bacteria, Tri C in eukaryotes):
  - GroEL: Formed of two independent folding chambers.
  - GroES: A cap that sequesters the protein inside the chamber during the ATP-dependent folding process.
  - The eukaryotic Tri C complex lacks a GroES homolog but operates similarly.

Binding and Enzymes

Antibodies (Immunoglobulins): Multimeric proteins consisting of two heavy chains and two light chains held by disulfide bridges. Binding occurs at the CDRs (Complementarity Determining Regions).
Enzyme Catalysis Principles:
- Enzymes speed up reaction rates by lowering the Activation Energy needed to reach the Transition State.
- Enzymes cannot alter the $\Delta G$ (change in free energy) or make non-spontaneous reactions occur.
- Active Site: Comprised of a Binding Pocket (for substrate specificity) and a Catalytic Site (where the chemical reaction occurs).
Serine Proteases: Enzymes like Trypsin, Chymotrypsin, and Elastase that cleave peptide bonds using a serine residue (e.g., Serine $195$ ). They differ in their binding pocket specificity (e.g., Trypsin uses Aspartate $189$ to recognize Arginine).

Enzyme Kinetics: Michaelis-Menten Model

$V_{max}$ : The maximum velocity/rate of a reaction when the enzyme is fully saturated with substrate.
$K_m$ (Michaelis Constant): The substrate concentration at which the reaction rate is exactly half of $V_{max}$ ( $0.5 \times V_{max}$ ).
- A lower $K_m$ indicates a higher affinity for the substrate.
Pathway Efficiency:
1. Diffusion: Discrete enzymes required to find products of previous steps via random motion; least efficient.
2. Scaffolds: Proteins that hold several enzymes in a pathway physically close for faster transfer.
3. Multifunctional Polypeptides: A single polypeptide containing multiple distinct catalytic activities; most efficient.

Regulation of Protein Activity

Quantity Regulation: Controlled by synthesis vs. degradation (life span ranging from minutes to the life of the cell).
Location Regulation: Transporting proteins to the nucleus, mitochondria, or other specific sites.
Non-Covalent Modification:
- Allostery/Cooperativity: Example: Hemoglobin's S-shaped oxygen binding curve. Binding of the first oxygen increases the affinity of the remaining subunits.
- Calmodulin: Changes from a dumbbell shape to a circular target-binding conformation upon binding four calcium ions non-covalently.
- GTP-Switch Proteins: Active when bound to GTP; inactive when bound to GDP. Their state is toggled by GEFs (Guanine Exchange Factors) and their own intrinsic GTPase activity.
Covalent Modification:
- Phosphorylation: Addition of phosphate by Kinases and removal by Phosphatases. Usually occurs on Serine, Threonine, or Tyrosine. It can either activate or deactivate a protein.
- Proteolytic Processing: Cleaving precursors into active forms (e.g., Zymogens into digestive enzymes; Prohormones into hormones; POMC cleavage into multiple distinct peptides).
Ubiquitination and The Proteasome:
- The Signaling Enzymes:
  - $E1$ : Ubiquitin Activating Enzyme.
  - $E2$ : Ubiquitin Conjugating Enzyme.
  - $E3$ : Ubiquitin Ligase (transfers Ub to target protein via an isopeptide bond).
- Proteasome Architecture: Consists of a $20S$ central core (contains proteases) and two $19S$ caps (recognize polyubiquitinated proteins, remove Ub, and unfold the target).
- Polyubiquitin Lysine Linkages:
  - Lysine $48$ : Signal for proteasomal degradation.
  - Lysine $63$ : Signal for immunity signaling.
  - Lysine $33$ : Control of T lymphocytes.
  - Lysine $11$ : Involved in cell division.