Week 10 HW: Imaging and Measurement

This page tackles all homeworks of week 10.

Homework: Final Project

  • Identify at least one (ideally many) aspect(s) of your project that you will measure. It could be the mass or sequence of a protein, the presence, absence, or quantity of a biomarker, etc.

    • For my final project investigating how protein synthesis direction changes a protein's structure for identical amino acid sequences, the core physical metrics I need to measure are: 1) Primary Structure Verification: Confirming that both the forward-synthesized protein (Ubiquitin/GB1) and the reverse-synthesized retro-protein contain the identical amino acid sequence but in inverted directions. 2) Tertiary Fold and Structural Topography: Measuring the actual 3D conformation of the retro-GB1 protein to see if it adopts a different stable fold than the highly structured wild-types. 3) Protein Purity and Mass: Verifying the molecular weight of the expressed variants to ensure full-length translation without premature truncation.

  • Please describe all of the elements you would like to measure, and furthermore describe how you will perform these measurements.

    1. Sequence Orientation & Intact Mass: Measure the exact molecular weight of the intact, full-length retro-protein chain. To confirm the directional sequence, the protein will be broken down into specific peptide fragments.
    2. Higher-Order Structure (Secondary/Tertiary Fold): Measure the atomic-scale spatial coordinates, secondary structures (beta-sheets, alpha-helices), and the overall 3D folded geometry of the crystallized retro-protein.
    3. Environmental/Gravitational Co-translational Dynamics: Monitor structural differences when the proteins are synthesized under normal Earth gravity versus a microgravity environment to isolate convective or gravitational confounding variables during co-translational folding.
    4. Temperature-dependent final state: Monitor the final protein structure if the stable temperature is changed across multiple experimental setups. This will also help understand how the temperature inside bodies can affect protein folding and whether climate change-induced global warming can affect final protein structures (temperature definitely affects the protein structure, but it is necessary to find when this effect starts to cause problems). Temperature-dependent protein structure differences for the same/retro sequence can be different for different proteins and for different homeostatic environments, and therefore, some specific proteins could be worse affected by global warming than others. This will also help identify those proteins which are more thermally stable for humans (and ultimately allow synthesis of the other proteins to evolve into more thermally stable stages) ~ heat-resistant humans!

  • What are the technologies you will use (e.g., gel electrophoresis, DNA sequencing, mass spectrometry, etc.)? Describe in detail.

    1. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): Use intact mass spectrometry to confirm the exact total mass of the expressed protein variants. Following this, I will perform bottom-up peptide mapping via tryptic digestion and MS/MS sequencing to read the exact primary sequence directionally, verifying the complete inversion of amino acid coordinates.
    2. X-ray Crystallography or Cryo-EM: To rigorously evaluate my core hypothesis that retro-variants fold into different structural topologies, I will use X-ray crystallography (or high-resolution Cryo-EM) to map out the explicit atomic-scale tertiary fold of retro-Ubiquitin. This provides the physical ground truth to compare directly against my low-pLDDT AlphaFold structural predictions.
    3. Space-based Cell-Free Expression Chambers: To test whether gravitational fields act as a confounding force during co-translational synthesis, I will use automated microfluidic cell-free expression systems running in microgravity environments (such as on the ISS) contrasted directly against identical Earth-bound control reactions.
    4. To understand temperature-dependent variations, many test setups need to be separated for protein synthesis in different temperature environments, both in cell-free systems and within organisms (Phytotrons are controlled-environment facilities designed to study growth and interactions with environmental factors under precisely regulated conditions).

Waters Homeworks

For these Homeworks:

  1. Waters Part I — Molecular Weight
  2. Waters Part II — Secondary/Tertiary structure
  3. Waters Part III — Peptide Mapping - primary structure
  4. Waters Part IV — Oligomers
  5. Waters Part V — Did I make GFP?


raw lab spectrum data sheets, peak lists, or observed m/z values generated from the Waters Immerse Lab equipment is necessary to calculate exact experimental molecular weights, isotopic charge states, and parts-per-million (ppm) mass errors! I did not attend any labs being a Global Committed Listener!
Imp. Note to self: Check all the related videos and see if you can actually ask for a grant to perform these experiements within IIT Kharagpur’s Central Research facility, or other places near Kolkata. Also check YoutTube tutorials and pages of fellow students who were able to complete this…