Week 10 HW: Imaging and Measurement

Final Project

For my final project, I will measure the binding affinity of my de novo peptide to the beta-gamma subunits of the human µ opioid receptor (MoR) using AlphaFold3. If I were to incorporate a wet lab component to this project, I would measure the GFP fluorescence of E. coli cells with my engineered plasmid (peptide+GFP) electroporated into them to confirm the genes of interest are being expressed then perform co-immunoprecipitation to measure the binding affinity of the de novo peptide to the human MoR Gβγ subunits.

Part I: Molecular Weight

1. The theoretical molecular weight of the eGFP protein (without the linker and histidine tag) is 26941.48 Da. With the linker and histidine tag, the molecular weight is 28006.60 Da.
2. Charge state approach:
   • z = 32.09
   • MW = 27823 Da
   • Accuracy –> 0.65% off from true value
3. Perhaps if I had a higher resolution image where I could more accurately read the peaks, but with the image provided, no.

Part II: Secondary/Tertiary Structure

1. Native state proteins are stabilized by intermolecular forces (i.e. hydrogen bonds, disulfide bonds, VDW interactions, hydrophobic interactions, etc) with well-ordered and low free energy (ΔG). Denaturated proteins are prone to aggregation (of hydrophobic regions) or protonation (of basic regions) as amino acid residues that would be insulated on the protein interior in the native state are exposed to the surrounding environment. A denatured protein will have more peaks than a native state protein due to greater numbers of charges.
2. The charge state appears to be around +4.6. This is calculated by z = 1/(2799.6365-2799.4199)

Part III: Peptide Mapping - Primary Structure

1. 20 lysine, 6 arginine
2. 19 peptides
3. Yes, the number of peaks (~20) is approximately the same as the number of peptides
4. One more peak than expected

Part IV: Oligomers

7FU Decamer: Intensity ~15 (at 3.4 MDa)

8FU Didecamer: Intensity ~170 (at 8 MDa)

8FU 3-Decamer: Intensity ~50 (at 12 MDa)

8FU 4-Decamer: Intensity close to 0 (at 16 MDa)

Part V: Did I Make GFP?

Our lab did not have work done at the Wates Immerse Lab, so this is the table from the screenshot data: