Notes on Protein Structure, Folding, and Dynamics (Lecture Summary)

Structure-Function Relationship in Proteins

The fundamental principle that the precise atomic arrangement and three-dimensional shape of a protein dictate its specific biological role. This applies universally across all protein types, from transcription factors to enzymes.

Levels of Protein Structure

Proteins have four levels of structure: 1. Primary (amino acid sequence). 2. Secondary (local folding patterns like alpha-helices and beta-sheets). 3. Tertiary (overall 3D shape of a single polypeptide chain). 4. Quaternary (assembly of multiple polypeptide subunits).

Amino Acid Basic Structure

Each standard amino acid has a central α-carbon, an amino group (H*{3}N^{+}), a carboxyl group (COO^{-}), a hydrogen atom, and a unique side chain (R-group). The N-terminus (α- ext{NH}_3{+}) has a pKa ~9.6, and the C-terminus (α- ext{COO}^{-})) has a pKa ~2.3.

R-group

The R-group (side chain) is the defining feature of each amino acid, dictating its full identity, chemical properties (e.g., hydrophobicity, charge, polarity, size), and specific reactivity within a protein.

Histidine's Role in Catalysis

Histidine's imidazole ring has an intrinsic pKa ~6.0, making it uniquely titratable near physiological pH. This tunable pKa allows it to act as both a proton donor and acceptor in enzyme active sites, central to many catalytic mechanisms.

Cysteine's Role in Catalysis

Cysteine's sulfhydryl (–SH) group can ionize to a thiolate (–S−), a potent nucleophile. Its intrinsic pKa (~8.3) can be significantly lowered (e.g., to 6–8) in an enzyme active site due to the microenvironment, thus increasing thiolate concentration and enhancing its nucleophilicity for catalysis.

Peptide Bond

An amide linkage formed via a condensation reaction between the carboxyl group of one amino acid and the amino group of the next, releasing water. It forms the primary structure, connecting amino acids in a linear sequence from N-terminus to C-terminus.

Peptide Bond Resonance and Planarity

The peptide bond exhibits partial double-bond character (approximately 40%) due to resonance, which restricts rotation around the C-N bond and confers planarity: the six atoms involved (αC1, C1, O1, N2, H2, αC2) lie rigidly in a single plane.

Cis/Trans Isomerism in Peptide Bonds

The ω (omega) torsion angle is typically 180^ ext{o} (trans configuration) to minimize steric clash. The cis configuration (0^ ext{o}) is rare, except for X-Pro peptide bonds (where up to 10-30% can be cis) due to proline’s unique cyclic structure reducing the steric difference.

Proline Isomerases

Specialized enzymes (e.g., Pin1) that catalyze the slow cis–trans interconversion of X-Pro peptide bonds. This isomerization can be a rate-limiting step in protein folding and is critical for regulating protein function.

Ramachandran Plot

A graphical map of the φ (N–C{ ext{α}}) vs. ψ (C{ ext{α}}–C) torsion angles for amino acid residues in a polypeptide chain. It reveals sterically allowed regions corresponding to common secondary structures (e.g., α-helices, β-sheets) and disallowed regions where atoms clash.

Alpha Helix

A common, rod-like, right-handed secondary structure stabilized by regular intra-strand hydrogen bonds between the carbonyl oxygen of residue i and the amide hydrogen of residue i+4. Side chains point outward, and the helix has a strong macroscopic dipole moment (N-terminus positive, C-terminus negative).

Beta Sheet

Composed of extended β-strands, where hydrogen bonds form between the backbone atoms of neighboring β-strands. Can be antiparallel (stronger, linear H-bonds) or parallel (weaker, bent H-bonds). Side chains alternate up and down, extending perpendicular to the pleated sheet.

Loops and Turns

Irregular, non-repeating segments connecting secondary structure elements. Turns are short, sharply defined (e.g., β-turns, 4 residues, H-bond i to i+3) that reverse chain direction. Loops are longer, more variable, and often flexible. Glycine and proline are enriched in these regions due to their flexibility and kinking properties.

Tertiary Structure

The complete three-dimensional arrangement of all atoms in a single polypeptide chain, including secondary structures, loops, motifs, and side chain positions. Stabilized by non-covalent interactions (hydrophobic, H-bonds, salt bridges, van der Waals) and often covalent disulfide bonds.

Protein Domains

Distinct, relatively compact, and often independently folding units within a single polypeptide chain. They frequently possess specific, self-contained functions (e.g., DNA binding, catalytic activity) and demonstrate modularity in protein architecture.

Quaternary Structure

The precise assembly and spatial arrangement of multiple polypeptide subunits (each with its own tertiary structure) into a larger, functional multi-subunit protein complex. Stabilized by non-covalent interactions and sometimes disulfide bonds between different subunits. Essential for cooperative functions, like hemoglobin's oxygen binding.

Hydrophobic Effect

The primary entropy-driven force for protein folding and stability. It involves the spontaneous burying of nonpolar amino acid side chains into a protein's core, releasing ordered water molecules (clathrates) back into bulk solvent, thereby significantly increasing the overall entropy of the solvent system. This entropy gain makes the folded state thermodynamically favorable.

Chaperones

Proteins that assist in proper protein folding by interacting with nascent or partially unfolded polypeptides. They lower energy barriers for folding steps, prevent misfolding into incorrect aggregates, and inhibit the aggregation of unfolded intermediates, especially under cellular stress.

Misfolding and Disease (CFTR ΔF508)

Misfolded proteins can expose hydrophobic regions, leading to toxic oligomers and insoluble aggregates linked to diseases (e.g., Alzheimer's). The CFTR ΔF508 deletion mutation causes a misfolded protein that fails to reach the cell membrane, impairing its function as a chloride channel and leading to cystic fibrosis symptoms.

Proteostasis

The intricate cellular network that maintains a healthy and functional population of proteins through coordinated processes of synthesis, folding, modification, transport, and degradation. Disruptions contribute to aging and diseases.

Stabilizing Interactions in Proteins

Include: 1. Hydrogen bonds (between backbone atoms or side chains). 2. Salt bridges (electrostatic interactions between oppositely charged ionic groups). 3. Disulfide bonds (covalent –S–S– links between two cysteines). 4. Hydrophobic interactions (burying nonpolar groups). Ordered water molecules can also stabilize protein structures.

Chaotropic Agents

Substances (e.g., urea, guanidinium chloride) that denature proteins by disrupting the hydrogen-bond network of bulk water and directly interacting with proteins, destabilizing their hydration shell and interfering with hydrophobic interactions, thereby promoting unfolding.

What is the core message regarding protein structure and function?

Structure dictates function: the precise atomic arrangement of a protein dictates its biological role.

What are the components of a standard amino acid?

A central α-carbon, an amino group (H*{3}N^{+}), a carboxyl group (COO^{-}), a hydrogen atom, and a unique side chain (R-group).

What is the significance of the R-group in amino acids?

It is the defining feature, dictating the amino acid's identity, chemical properties (hydrophobicity, charge, polarity, size), and specific reactivity.

Why is Histidine unique among basic amino acids?

Its imidazole ring has an intrinsic pKa around 6.0, making it uniquely titratable near physiological pH, allowing it to act as both a proton donor and acceptor in enzyme active sites.

How does the microenvironment affect Cysteine's reactivity?

The local microenvironment in an enzyme active site can significantly lower cysteine's intrinsic pKa (from ~8.3 to 6-8), increasing the concentration of its potent nucleophilic thiolate (–S−) form, thereby boosting catalytic efficiency.

What is primary protein structure?

The linear, unique sequence of amino acids connected by peptide bonds, genetically encoded.

Describe a peptide bond.

An amide linkage formed via a condensation reaction between the carboxyl group of one amino acid and the amino group of the next, releasing water. It has partial double-bond character and planarity, restricting rotation.

Why is the peptide bond planar?

It exhibits significant partial double-bond character due to resonance stabilization, which severely restricts rotation around the C-N bond, causing the six participating atoms to lie rigidly in a single plane.

What are the allowed torsion angles in a polypeptide backbone?

Rotation is only possible around the N–$C{ ext{α}}$ (φ, phi) and $C{ ext{α}}$–C (ψ, psi) bonds, not the peptide bond itself (ω, omega).

What is the favored configuration for peptide bonds?

The trans configuration ( ext{ω} ext{ approximately } 180^ ext{o}) is overwhelmingly favored for most amino acids to minimize steric clash between R-groups and the carbonyl oxygen.

When can a cis peptide bond occur significantly?

X-Pro peptide bonds (where X is any amino acid preceding Proline) can adopt cis or trans configurations with significant frequency (10-30% cis) due to proline's unique cyclic structure reducing the steric difference.

What is the function of Proline isomerases?

They catalyze the relatively slow cis–trans interconversion of X-Pro peptide bonds, a rate-limiting step in protein folding and critical for regulating protein function.

What does a Ramachandran plot show?

It graphically maps the φ vs. ψ torsion angles for all residues in a protein, revealing sterically allowed regions corresponding to common secondary structures and disallowed regions where atoms would clash.

Why is Glycine more flexible on a Ramachandran plot?

Its R-group is just a hydrogen atom, lacking a bulky side chain, allowing it to adopt a wider range of φ/ψ values and making it highly flexible.

What are alpha-helices?

Common, rod-like, right-handed helical structures principally stabilized by intra-strand hydrogen bonds between the carbonyl oxygen of residue i and the amide hydrogen of residue i+4.

What are beta-sheets?

Composed of extended β-strands, forming sheet-like structures stabilized by hydrogen bonds between the backbone atoms of neighboring β-strands (either antiparallel or parallel).

Contrast antiparallel and parallel β-sheets.

Antiparallel β-sheets have neighboring strands running in opposite directions, allowing for stronger, more linear hydrogen bonds. Parallel β-sheets have strands running in the same direction, resulting in slightly weaker, bent hydrogen bonds.

What are loops and turns?

Irregular, non-repeating segments that connect secondary structure elements, crucial for reversing polypeptide chain direction and forming active sites. Turns are short (e.g., β-turns, 4 residues); loops are longer and more variable.

Why are Glycine and Proline enriched in loops and turns?

Glycine's flexibility allows for sharp bends, while proline's rigid cyclic structure readily induces kinks and turns, facilitating direction changes.

What is tertiary protein structure?

The complete three-dimensional arrangement of all atoms in a single polypeptide chain, stabilized by non-covalent interactions (hydrophobic, hydrogen bonds, salt bridges, van der Waals) and often disulfide bonds.

What are protein domains?

Distinct, relatively compact, and often independently folding units within a single polypeptide chain, frequently possessing specific, self-contained functions.

What is quaternary protein structure?

The precise assembly and spatial arrangement of multiple polypeptide subunits into a larger, functional multi-subunit protein complex, stabilized by non-covalent interactions and sometimes disulfide bonds between subunits.

What is the primary driving force for protein folding and stability?

The hydrophobic effect. It is an entropy-driven phenomenon where the burying of nonpolar residues in the protein core releases ordered water molecules (clathrates) back into bulk solvent, increasing overall solvent entropy.

What are chaperones in protein folding?

Proteins that assist in proper protein folding by interacting with nascent or partially unfolded polypeptides, preventing misfolding into incorrect aggregates and inhibiting aggregation, especially under cellular stress.

How does the CFTR deltaF508 mutation cause cystic fibrosis?

A single deletion of phenylalanine at position 508 results in a misfolded CFTR protein, preventing it from correctly reaching the cell membrane, thus impairing its function as a chloride channel.