Melany Jumbo, Quito, Ecuador 🇪🇨

Objective:

1. Learn basic concepts: amino acid structure, 3D protein visualization, and the variety of ML-based design tools.
2. Brainstorm as a group how to apply these tools to engineer a better bacteriophage (setting the stage for the final project).

Part A. Conceptual Questions

• Answer any of the following questions by Shuguang Zhang:

  • 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)

  A: 100g Meat —> 26g protein

  500g Meat —> X

  X = (500g M * 26g P)/ (100g M) = 130g of Protein Aprox.

  According Waters (s.f.)= Average weight of aminoacid is 100Da (Dalton or g/mol)

  = 130g/ (100g/mol) = 1.18 mol

  = 1.3 * 6.022x10^23 (Avogadro’s)

  = 7.8286x10^23 molecules of aa.

  Reference: https://fdc.nal.usda.gov/food-details/168250/nutrients

  • Why humans eat beef but do not become a cow, eat fish but do not become fish?

  A: By breaking down protein into amino acids. This is related to diet, rather what determines whether a certain living being has plant or animal cells is DNA, the genetic material is the only one that can code for specific cells for the different parts of the cow and so on with each animal.

  • Why there are only 20 natural amino acids?

  A: Due to the ability of the ribosome to synthesize proteins and the chemical structure of nucleotides.

  • Can you make other non-natural amino acids? Design some new amino acids.

  A: Yes, non-natural amino acids can be created through chemical synthesis. For example, amino acids can be designed with specific properties to improve protein stability or activity.

  • Where did amino acids come from before enzymes that make them, and before life started?

  A: If I'm not mistaken, there are a few theories, but based on research, amino acids were probably formed on early Earth through inorganic chemical reactions, such as those described in the Miller-Urey experiment. These reactions generated organic compounds, including amino acids, from atmospheric gases and energy.

  • If you make an alpha-helix using D-amino acids, what handedness (right or left) would you expect?

  A: Natural alpha-helices are made from L-amino acids and are generally left-handed helices. However, if an alpha-helix were built from D-amino acids, it would be expected to be a right-handed helix due to the reversal of chirality.

  • Can you discover additional helices in proteins?

  A: Yes, there are pi helices for example that are not very typical like alpha and beta helices, but they are important for certain types of proteins.

  • Why most molecular helices are right-handed?

  A: Because of the chilarity of L-amino acids. Since it gives more stability due to the hydrogens. Sometimes the chemical position in which certain elements are found can make a big difference.

  • Why do beta-sheets tend to aggregate?

  A: It may be due to irregularities in the amino acid sequence and hydrogen bonding patterns in the observed sheets, or to long-range interactions in proteins.

  • What is the driving force for b-sheet aggregation?

  A: Hydrofobic interactions and Van der Waals forces

  • Why many amyloid diseases form b-sheet?

  A: This is because certain proteins can adopt disordered beta structures that aggregate and form stable fibrils.

  • Can you use amyloid b-sheets as materials?

  A: Yes, amyloid structures can be used as materials due to their stability and unique mechanical properties.

  • Design a b-sheet motif that forms a well-ordered structure.

  A:

  • Sequence

PyMOL - Author Design

Part B: Protein Analysis and Visualization

In this part of the homework, you will be using online resources and 3D visualization software to answer questions about proteins.

1. Pick any protein (from any organism) of your interest that has a 3D structure and answer the following questions.

Myoglobin proteín -3D

• Briefly describe the protein you selected and why you selected it.

A: Myoglobin is a muscle protein that has a mixed structure, meaning it has half of it that is protein and half that is not. It is considered a heme protein. I like it because it is one of the most studied proteins and there is a lot of information about it. In addition, it is associated with damage to the body since it is found in skeletal and heart muscles.

• Identify the amino acid sequence of your protein.

Important: Myoglobin is a water-soluble protein composed of a single polypeptide chain, globin, containing eight α-helices.

• How long is it? What is the most frequent amino acid? You can use this notebook to count most frequent amino acid - https://colab.research.google.com/drive/1vlAU_Y84lb04e4Nnaf1axU8nQA6_QBP1?usp=sharing

A: It has 150 from 154 aa, according to the DNA instructions. The mos frerquent amino acid are leucine or glutamine.

• How many protein sequence homologs are there for your protein?Hint: Use the pBLAST tool to search for homologs and ClustalOmega to align and visualize them. Tutorial Here

• A: There are a lot of homologs, >100 of the ones I could count.

• Does your protein belong to any protein family?

A: It belongs to the globin family.

REFERENCE: https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/myoglobin, https://blast.ncbi.nlm.nih.gov/Blast.cgi# , https://www.ncbi.nlm.nih.gov/protein/CAA25109.1?report=fasta

• Identify the structure page of your protein in RCSB

  • When was the structure solved? Is it a good quality structure? Good quality structure is the one with good resolution. Smaller the better (Resolution: 2.70 Å)

  A: https://www.rcsb.org/3d-view/9BA2 (2024) It has a good resolution = 2.97 Angstrom

  

  • Are there any other molecules in the solved structure apart from protein?

  A: Yes, like an heme group.

  • Does your protein belong to any structure classification family?

  A:

  Class: All alpha Family: globins Superfamily: Globin-like Protein domain: globin

• Open the structure of your protein in any 3D molecule visualization software:

  • PyMol Tutorial Here (hint: ChatGPT is good at PyMol commands)
  • Visualize the protein as "cartoon", "ribbon" and "ball and stick".
  • Color the protein by secondary structure. Does it have more helices or sheets?

  It has more Alpha Helices (connected by loops) than Beta Helices

  • Color the protein by residue type. What can you tell about the distribution of hydrophobic vs hydrophilic residues?

  Hydrophobic Residues: Look for residues like Alanine (A), Valine (V), Leucine (L), Isoleucine (I), Methionine (M), Phenylalanine (F), and Tryptophan (W).

  Hydrophilic Residues: Look for residues like Lysine (K), Arginine (R), Histidine (H), Aspartic Acid (D), Glutamic Acid (E), Asparagine (N), and Glutamine (Q).

  • Visualize the surface of the protein. Does it have any "holes" (aka binding pocke

Helix (Sky blue), Sheet (purple) and pink (loop).

Part C. Using ML-Based Protein Protein Tools