Week 4: Protein Design

Assignment 4: Protein Engineering

Part A: Conceptual Questions

1. How many molecules of amino acids do you take with a piece of 500 grams of meat? (on average an amino acid is ~100 Daltons) To calculate this, we use a back-of-the-envelope estimation. Meat is not purely protein; it consists mostly of water and fat. Assuming meat contains about 20% protein by weight, 500 grams of meat yields roughly 100 grams of pure protein. By definition, 1 gram is approximately equal to Avogadro’s number in Daltons (6.022 x 1023 Da). Therefore, 100 grams of protein equals 6.022 x 1025 Daltons. Dividing this total mass by the average mass of a single amino acid (100 Daltons) gives 6.022 x 10^23 molecules. Fascinatingly, eating 500 grams of meat equates to consuming almost exactly 1 mole of amino acids.

2. Why do humans eat beef but do not become a cow, eat fish but do not become fish? The answer lies in the difference between “building blocks” and “blueprints.” When a human eats beef, the digestive system breaks down the cow’s complex proteins completely into individual amino acids. These amino acids are universal building blocks devoid of the cow’s genetic information. Once absorbed into the bloodstream, human cells use their own DNA blueprint and ribosomes to reassemble these generic amino acids into human-specific proteins.

3. Why are there only 20 natural amino acids? This is an evolutionary compromise between chemical sufficiency and metabolic burden. These 20 amino acids provide a complete chemical toolkit (including polar, non-polar, acidic, basic, and aromatic side chains) sufficient to fold into complex 3D structures and catalyze almost any required biochemical reaction. Evolving to support more amino acids would require maintaining additional tRNAs and synthetases, increasing the metabolic cost. Furthermore, assigning 61 codons to just 20 amino acids creates a highly degenerate genetic code, making life significantly more robust against lethal DNA mutations.

4. Can you make other non-natural amino acids? Design some new amino acids. Yes, through Genetic Code Expansion. By hijacking a stop codon (usually the Amber codon, UAG) and introducing an orthogonal tRNA/synthetase pair into the cell, we can incorporate unnatural amino acids (uAAs) into proteins.

  • Design 1: Click-Amino Acid. An amino acid carrying an Azide group (-N3) on its side chain. Since azides are biologically inert, we can use “Click Chemistry” to precisely attach fluorescent tags or drugs to this specific residue after the protein is translated.
  • Design 2: Photo-switchable Amino Acid. An amino acid featuring an Azobenzene group. Azobenzene undergoes a structural shift (from trans to cis) when exposed to UV light. Placing this in an enzyme’s active site would create a biological machine that can be turned on and off with light.

5. Where did amino acids come from before enzymes that make them, and before life started? Amino acids are thermodynamically stable molecules that can synthesize spontaneously from simple chemical precursors without enzymes (abiogenesis). The famous 1953 Miller-Urey experiment demonstrated that applying electrical sparks (simulating lightning) to a mixture of early Earth gases (methane, ammonia, hydrogen, and water vapor) naturally produces amino acids like glycine and alanine. Additionally, astrobiology has shown that amino acids form in deep space via cosmic radiation; they have been abundantly found in carbonaceous meteorites, such as the Murchison meteorite.

6. If you make an α-helix using D-amino acids, what handedness (right or left) would you expect? We would expect a left-handed helix. Natural proteins use L-amino acids, which fold into right-handed helices to minimize steric hindrance (physical clashing) between their side chains. Because D-amino acids are exact mirror images (enantiomers) of L-amino acids, the spatial forces are reversed. To achieve the lowest energy state and avoid steric clashes, a chain of D-amino acids must coil in the exact opposite direction, resulting in a left-handed helix.

7. Can you discover additional helices in proteins? Yes, while the alpha-helix is the most common, other helical structures exist based on different hydrogen-bonding patterns:

  • 3_10-helix: A tighter, more elongated helix where the hydrogen bond forms between residue i and i+3.
  • Pi-helix (π-helix): A wider, more compressed helix with hydrogen bonds between residue i and i+5. These are often found at enzyme active sites due to their functional flexibility.
  • Polyproline helix: Formed by consecutive proline residues, which act as “helix breakers” for standard alpha-helices. It lacks intra-chain hydrogen bonds and forms an extended, left-handed helix (Type II) critical for structural proteins like collagen.

8. Why are most molecular helices right-handed? The preference for right-handed alpha-helices in nature is dictated by the chirality of L-amino acids and the rules of thermodynamics. When L-amino acids form a right-handed coil, their bulky side chains (R-groups) point outward and slightly downward. This orientation maximizes the distance between the side chains and the peptide backbone, minimizing steric hindrance. Forcing L-amino acids into a left-handed helix pushes the side chains too close to the backbone’s carbonyl oxygens, causing severe electron repulsion and energetic instability.

9. Why do β-sheets tend to aggregate? What is the driving force for β-sheet aggregation? Unlike alpha-helices, which satisfy all their hydrogen-bonding requirements internally, beta-sheets have exposed, “sticky” edges. The outermost strands of a beta-sheet possess unsatisfied hydrogen bond donors (N-H) and acceptors (C=O) that readily bind to adjacent beta-strands. The primary driving force is the hydrophobic effect; the hydrophobic faces of different beta-sheets come together to escape water. Once physically close, a massive intermolecular hydrogen-bonding network forms between the backbones. This cooperative bonding locks the sheets together in a deep global energy minimum, making the aggregate incredibly stable.

10. Why do many amyloid diseases form β-sheets? Can you use amyloid β-sheets as materials? In amyloid diseases (like Alzheimer’s), misfolded proteins expose their hydrophobic cores. To escape the aqueous cellular environment, these proteins aggregate. Thermodynamics dictates that the most stable conformation for this aggregation is the “cross-beta spine”—a highly ordered stacking of beta-sheets. This structure is so tightly bonded that cellular proteases cannot degrade it, leading to toxic plaque accumulation. Yes, we can use engineered (non-toxic) amyloids as biomaterials. Because of their extreme mechanical strength (comparable to steel or spider silk) and spontaneous self-assembly, scientists use amyloid fibrils to create highly durable hydrogels for tissue engineering, drug delivery systems, and biological nanowires.

11. Design a β-sheet motif that forms a well-ordered structure. To create a self-assembling, well-ordered beta-sheet, we must design an alternating sequence of hydrophobic and hydrophilic/charged residues.

  • Motif Example: (AEAEAKAKAEAEAKAK)
  • (A = Alanine [hydrophobic], E = Glutamic acid [negative], K = Lysine [positive])
  • Mechanism: When this peptide folds into a beta-strand, all the hydrophobic Alanines face one side, while the charged E and K residues face the opposite side. The hydrophobic faces from different sheets pack tightly together to exclude water. On the hydrophilic side, the alternating positive (K) and negative (E) charges create precise ionic salt bridges with neighboring strands. This electrostatic “zipper” forces the beta-sheets to align with nanometer-level precision, forming a highly ordered and structurally rigid scaffold.