Week 4 HW: Protein Design Part I

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)

500g of meat = ~20% protein 100g ÷ 110 g/mol ≈ 0.91 mol 0.91 × 6.022 × 10²³ ≈ 5.5 × 10²³ molecules*

Why are there only 20 natural amino acids?

Because of the frozen accident!! https://pubmed.ncbi.nlm.nih.gov/27926995/ Evolution has developed so, and it’s too late to change that also, they have the optimal protein structure

Where did amino acids come from before enzymes that make them, and before life started? Based on this Nature article( https://www.nature.com/scitable/topicpage/an-evolutionary-perspective-on-amino-acids-14568445/ ) from metabolic pathways up to Archean Eon and chemical synthesis up to Hadean Eon

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

Can you discover additional helices in proteins? Yes, through like DSSP or PROCHECK software.

Why are most molecular helices right-handed? They’re more stable! https://www.ch.ic.ac.uk/rzepa/blog/?p=3802

Why do β-sheets tend to aggregate? their structure has edge strands with hydrogen-bonding capacity, making them thermodynamically prone to stacking through edge-to-edge or face-to-face interactions

What is the driving force for β-sheet aggregation?

It is determined by ATR-FTIR spectroscopy

Why do many amyloid diseases form β-sheets?

“Amyloid beta peptide (Aβ) is produced through the proteolytic processing of a transmembrane protein, amyloid precursor protein (APP), by β- and γ-secretases. Aβ accumulation in the brain is proposed to be an early toxic event”. https://www.nature.com/articles/aps201728

Can you use amyloid β-sheets as materials? Design a β-sheet motif that forms a well-ordered structure. Yes, they can be used as stable nano materials with applications in environmental sciences, material engineering, and translational medicines https://pmc.ncbi.nlm.nih.gov/articles/PMC8508955/. Still unsure how to design it

Part B: Protein Analysis and Visualization

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

I’ve selected LuxR as it controls bioluminescence through quorum sensing which is a “is the regulation of gene expression in response to fluctuations in cell-population density”(https://pubmed.ncbi.nlm.nih.gov/11544353/). As I want to do project in connection to CA it seemed fitting. For structure visualizatio I will be using TraR (PDB: 1L3L)

2. Identify the amino acid sequence of your protein.

from collections import Counter: Protein sequence provided by the user protein_sequence = “MSSNIESLYRQMLNEIIEQMEKEGKISREEALAVLESKLKKSNVSRDIILSHGFNSGVTQSAQPSSFLNNMAAIAKQNLHFSDIFTDLENQFIAELEELEQSYKNLVTKLSQKLDLSSLTEQALVELRSYLNQIKQSGLLDSAQNIFNFSEQISALQEAIGQLSQSIANLELQSREISELNQRVQDLEQELSFLSQQLASAQTQASQLQAQINQLNHQLEALEKAHQEALSRAKQEIEKLRS”

The length of the protein is: 242 aminoacids. The most common amino acid is: L, which appears 36 times.

How many protein sequence homologs are there for your protein? Hint: Use Uniprot’s BLAST tool to search for homologs. 250!

*The UniProt BLAST search identified over 250 homologs with highly significant E-values (ranging from ~0 to 10⁻²⁴), indicating LuxR is part of a large, well-conserved protein family. LuxR belongs to the LuxR-FixJ superfamily and the LuxR family of transcriptional regulators, which are widespread across gram-negative bacteria and typically involved in quorum sensing and gene regulation.

LuxR belongs to the TetR family RCSB PDB of transcriptional regulators, specifically to Vibrio alginolyticus variant, and more broadly to LuxR-FixJ

3. Identify the structure page of your protein in RCSB

https://www.uniprot.org/uniprotkb/P12746/entry#sequences

Resolution: 1.66 Å

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

Yes! LAE (3-OXO-OCTANOIC ACID (2-OXO-TETRAHYDRO-FURAN-3-YL)-AMIDE) and MSE (L-PEPTIDE LINKING)

Does your protein belong to any structure classification family?

Yes! https://www.uniprot.org/uniprotkb/P33905/entry#family_and_domains

LuxR (TraR) belongs to the autoinducer-regulated transcriptional regulatory protein family. The 2 domains are an N-terminal autoinducer-binding domain (Pfam: Autoind_bind) that binds the AHL quorum sensing signal, and a C-terminal HTH LuxR-type DNA-binding domain (Pfam: GerE) that activates transcription. Also classified under the LuxR-FixJ superfamily (SUPFAM) and the Winged Helix DNA-binding superfamily for its C-terminal domain.

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

:The structure is predominantly alpha-helical.

Hydrophobic residues are concentrated in the protein core, forming the structural interior and lining the AHL ligand-binding pocket.

It does have binding pockets

Part C. Using ML-Based Protein Design Tools

I also asked Gemini to identify mutation hotspots based on the compiled data in the heatmap:**

“Let’s analyze the mutation scan heatmap to identify potential mutation hotspots. I’ll write some code to scan through each position in the protein sequence and highlight those where several different amino acid substitutions are predicted to be favorable (meaning they have a high positive Log-Likelihood Ratio). Here’s the analysis:

Mutation Hotspots Identified (positions with at least 3 mutations having LLR > 0.5):

Position 2 (Original: Q):

Mutate to M: LLR = 7.67
Mutate to A: LLR = 2.68
Mutate to L: LLR = 2.55
Mutate to V: LLR = 2.43
Mutate to R: LLR = 2.33
Mutate to E: LLR = 2.02
Mutate to I: LLR = 2.00
Mutate to T: LLR = 1.99
Mutate to S: LLR = 1.91
Mutate to K: LLR = 1.90
Mutate to Y: LLR = 1.89
Mutate to F: LLR = 1.69
Mutate to G: LLR = 1.59
Mutate to Q: LLR = 1.59
Mutate to H: LLR = 1.33
Mutate to D: LLR = 1.31
Mutate to N: LLR = 1.26
Mutate to P: LLR = 1.16 Position 4 (Original: W):
Mutate to A: LLR = 1.31
Mutate to E: LLR = 1.11
Mutate to T: LLR = 1.07
Mutate to S: LLR = 1.01
Mutate to R: LLR = 1.00
Mutate to Q: LLR = 0.96
Mutate to D: LLR = 0.95
Mutate to K: LLR = 0.80 Position 5 (Original: L):
Mutate to L: LLR = 2.45
Mutate to I: LLR = 1.99
Mutate to A: LLR = 1.96
Mutate to V: LLR = 1.77
Mutate to T: LLR = 1.17
Mutate to F: LLR = 1.07
Mutate to R: LLR = 0.80
Mutate to Y: LLR = 0.76
Mutate to E: LLR = 0.72
Mutate to S: LLR = 0.56 Position 15 (Original: E):
Mutate to A: LLR = 1.13
Mutate to S: LLR = 1.00
Mutate to T: LLR = 0.62
Mutate to R: LLR = 0.62
Mutate to L: LLR = 0.58 Position 20 (Original: I):
Mutate to E: LLR = 5.70
Mutate to A: LLR = 5.51
Mutate to D: LLR = 5.31
Mutate to T: LLR = 4.86
Mutate to Q: LLR = 4.72
Mutate to S: LLR = 4.71
Mutate to R: LLR = 4.31
Mutate to K: LLR = 4.31
Mutate to G: LLR = 4.15
Mutate to N: LLR = 4.03
Mutate to P: LLR = 3.68
Mutate to V: LLR = 3.55
Mutate to L: LLR = 3.52
Mutate to H: LLR = 3.47
Mutate to I: LLR = 2.68
Mutate to M: LLR = 2.11
Mutate to F: LLR = 2.08
Mutate to Y: LLR = 1.95
Mutate to W: LLR = 1.11 Position 25 (Original: L):
Mutate to A: LLR = 3.12
Mutate to V: LLR = 2.93
Mutate to L: LLR = 2.73
Mutate to I: LLR = 2.70
Mutate to T: LLR = 1.85
Mutate to M: LLR = 1.40
Mutate to F: LLR = 1.15
Mutate to C: LLR = 0.80 Position 28 (Original: I):
Mutate to E: LLR = 0.99
Mutate to A: LLR = 0.73
Mutate to L: LLR = 0.64 Position 38 (Original: A):
Mutate to V: LLR = 1.76
Mutate to A: LLR = 1.56
Mutate to I: LLR = 1.23
Mutate to S: LLR = 0.90
Mutate to L: LLR = 0.81
Mutate to G: LLR = 0.79
Mutate to T: LLR = 0.53 Position 40 (Original: L):
Mutate to I: LLR = 1.66
Mutate to L: LLR = 1.64
Mutate to V: LLR = 1.53
Mutate to F: LLR = 0.63 Position 42 (Original: I):
Mutate to D: LLR = 2.31
Mutate to E: LLR = 1.39
Mutate to L: LLR = 1.08
Mutate to S: LLR = 0.71
Mutate to G: LLR = 0.63
Mutate to N: LLR = 0.62
Mutate to V: LLR = 0.62
Mutate to T: LLR = 0.60 Position 43 (Original: Q):
Mutate to D: LLR = 4.50
Mutate to E: LLR = 2.76
Mutate to N: LLR = 2.38
Mutate to P: LLR = 2.35
Mutate to S: LLR = 2.06
Mutate to T: LLR = 1.76
Mutate to A: LLR = 1.44
Mutate to G: LLR = 1.35
Mutate to H: LLR = 1.29
Mutate to R: LLR = 1.26
Mutate to Q: LLR = 1.06
Mutate to K: LLR = 0.92
Mutate to L: LLR = 0.80
Mutate to V: LLR = 0.51 Position 44 (Original: H):
Mutate to D: LLR = 1.89
Mutate to E: LLR = 1.59
Mutate to P: LLR = 1.31
Mutate to G: LLR = 0.72
Mutate to A: LLR = 0.55 Position 45 (Original: R):
Mutate to D: LLR = 2.75
Mutate to N: LLR = 1.61
Mutate to E: LLR = 1.59
Mutate to G: LLR = 1.42
Mutate to S: LLR = 1.10
Mutate to T: LLR = 0.62 Position 46 (Original: H):
Mutate to D: LLR = 1.93
Mutate to G: LLR = 1.62
Mutate to E: LLR = 1.26
Mutate to N: LLR = 0.61 Position 47 (Original: I):
Mutate to E: LLR = 1.81
Mutate to D: LLR = 1.51
Mutate to R: LLR = 1.04
Mutate to T: LLR = 0.98
Mutate to S: LLR = 0.90
Mutate to G: LLR = 0.88
Mutate to Q: LLR = 0.77
Mutate to K: LLR = 0.74
Mutate to A: LLR = 0.60 Position 48 (Original: T):
Mutate to G: LLR = 1.72
Mutate to L: LLR = 1.12
Mutate to D: LLR = 0.98
Mutate to E: LLR = 0.85
Mutate to A: LLR = 0.75
Mutate to R: LLR = 0.50 Position 53 (Original: Y):
Mutate to G: LLR = 1.56
Mutate to E: LLR = 0.93
Mutate to A: LLR = 0.90
Mutate to D: LLR = 0.89
Mutate to S: LLR = 0.68 Position 55 (Original: R):
Mutate to G: LLR = 1.31
Mutate to S: LLR = 1.27
Mutate to D: LLR = 1.14
Mutate to T: LLR = 1.12
Mutate to P: LLR = 0.75
Mutate to A: LLR = 0.56 Position 56 (Original: Q):
Mutate to P: LLR = 1.07
Mutate to G: LLR = 0.79
Mutate to D: LLR = 0.77
Mutate to E: LLR = 0.59 Position 57 (Original: W):
Mutate to D: LLR = 1.10
Mutate to G: LLR = 0.99
Mutate to E: LLR = 0.93
Mutate to S: LLR = 0.56 Position 58 (Original: Q):
Mutate to G: LLR = 2.29
Mutate to D: LLR = 1.82
Mutate to E: LLR = 1.73
Mutate to P: LLR = 1.65
Mutate to A: LLR = 1.52
Mutate to S: LLR = 1.45
Mutate to T: LLR = 1.40
Mutate to L: LLR = 1.33
Mutate to R: LLR = 1.20
Mutate to N: LLR = 1.14
Mutate to Q: LLR = 1.01
Mutate to K: LLR = 0.96
Mutate to H: LLR = 0.91
Mutate to V: LLR = 0.90
Mutate to I: LLR = 0.84
Mutate to F: LLR = 0.60
Mutate to Y: LLR = 0.57 Position 59 (Original: S):
Mutate to G: LLR = 1.33
Mutate to L: LLR = 1.02
Mutate to A: LLR = 0.89
Mutate to D: LLR = 0.89
Mutate to S: LLR = 0.84
Mutate to P: LLR = 0.82
Mutate to T: LLR = 0.63
Mutate to E: LLR = 0.61
Mutate to I: LLR = 0.53 Position 61 (Original: Y):
Mutate to E: LLR = 0.68
Mutate to D: LLR = 0.61
Mutate to A: LLR = 0.60 Position 63 (Original: D):
Mutate to D: LLR = 1.12
Mutate to S: LLR = 0.94
Mutate to A: LLR = 0.84
Mutate to G: LLR = 0.75
Mutate to P: LLR = 0.74
Mutate to T: LLR = 0.67
Mutate to L: LLR = 0.59
Mutate to E: LLR = 0.56 Position 65 (Original: K):
Mutate to A: LLR = 0.89
Mutate to P: LLR = 0.88
Mutate to E: LLR = 0.87
Mutate to L: LLR = 0.85
Mutate to G: LLR = 0.80
Mutate to D: LLR = 0.67
Mutate to R: LLR = 0.65 Position 66 (Original: F):
Mutate to S: LLR = 1.46
Mutate to P: LLR = 1.41
Mutate to D: LLR = 1.29
Mutate to T: LLR = 1.27
Mutate to G: LLR = 0.86
Mutate to A: LLR = 0.79
Mutate to R: LLR = 0.78
Mutate to E: LLR = 0.75 Position 75 (Original: K):
Mutate to A: LLR = 1.30
Mutate to E: LLR = 1.23
Mutate to L: LLR = 1.01
Mutate to R: LLR = 0.86
Mutate to D: LLR = 0.65
Mutate to S: LLR = 0.59
Mutate to W: LLR = 0.52 Position 78 (Original: R):
Mutate to L: LLR = 2.00
Mutate to I: LLR = 1.32
Mutate to F: LLR = 0.60
Mutate to V: LLR = 0.56 Position 80 (Original: R):
Mutate to G: LLR = 1.42
Mutate to E: LLR = 0.95
Mutate to D: LLR = 0.84
Mutate to S: LLR = 0.69
Mutate to A: LLR = 0.64 Position 82 (Original: H):
Mutate to G: LLR = 1.36
Mutate to E: LLR = 1.11
Mutate to D: LLR = 1.00
Mutate to T: LLR = 0.96
Mutate to P: LLR = 0.96
Mutate to S: LLR = 0.92
Mutate to R: LLR = 0.89
Mutate to A: LLR = 0.87
Mutate to L: LLR = 0.55 Position 84 (Original: F):
Mutate to L: LLR = 1.07
Mutate to V: LLR = 1.05
Mutate to I: LLR = 0.99 Position 85 (Original: T):
Mutate to V: LLR = 0.84
Mutate to L: LLR = 0.65
Mutate to I: LLR = 0.54 Position 86 (Original: W):
Mutate to V: LLR = 2.01
Mutate to L: LLR = 1.91
Mutate to I: LLR = 1.90
Mutate to A: LLR = 1.64
Mutate to T: LLR = 1.52
Mutate to S: LLR = 1.39
Mutate to R: LLR = 1.12
Mutate to F: LLR = 1.10
Mutate to E: LLR = 0.90
Mutate to D: LLR = 0.82
Mutate to G: LLR = 0.71
Mutate to Y: LLR = 0.63
Mutate to K: LLR = 0.62
Mutate to H: LLR = 0.62
Mutate to P: LLR = 0.56
Mutate to N: LLR = 0.53 Position 90 (Original: H):
Mutate to L: LLR = 1.31
Mutate to I: LLR = 1.28
Mutate to V: LLR = 1.09
Mutate to A: LLR = 1.03
Mutate to T: LLR = 0.94
Mutate to P: LLR = 0.79
Mutate to S: LLR = 0.76
Mutate to G: LLR = 0.75
Mutate to D: LLR = 0.64
Mutate to E: LLR = 0.61 Position 92 (Original: R):
Mutate to L: LLR = 1.41
Mutate to I: LLR = 0.92
Mutate to D: LLR = 0.91
Mutate to V: LLR = 0.67 Position 93 (Original: P):
Mutate to L: LLR = 1.17
Mutate to I: LLR = 0.76
Mutate to V: LLR = 0.73 Position 97 (Original: K):
Mutate to A: LLR = 1.41
Mutate to P: LLR = 1.39
Mutate to E: LLR = 0.93
Mutate to D: LLR = 0.80 Position 105 (Original: H):
Mutate to L: LLR = 1.84
Mutate to A: LLR = 1.60
Mutate to I: LLR = 1.40
Mutate to F: LLR = 1.26
Mutate to V: LLR = 1.24
Mutate to E: LLR = 0.89
Mutate to Y: LLR = 0.75
Mutate to T: LLR = 0.72
Mutate to R: LLR = 0.71 Position 107 (Original: S):
Mutate to L: LLR = 1.67
Mutate to R: LLR = 1.33
Mutate to I: LLR = 1.30
Mutate to V: LLR = 1.14
Mutate to A: LLR = 0.70 Position 108 (Original: D):
Mutate to R: LLR = 1.01
Mutate to A: LLR = 0.62
Mutate to E: LLR = 0.57
Mutate to K: LLR = 0.51 Position 118 (Original: P):
Mutate to A: LLR = 0.97
Mutate to G: LLR = 0.81
Mutate to R: LLR = 0.67
Mutate to L: LLR = 0.50 Position 123 (Original: N):
Mutate to D: LLR = 1.15
Mutate to T: LLR = 0.76
Mutate to S: LLR = 0.58 Position 125 (Original: F):
Mutate to E: LLR = 1.39
Mutate to V: LLR = 1.08
Mutate to R: LLR = 1.00
Mutate to L: LLR = 0.98
Mutate to K: LLR = 0.93
Mutate to I: LLR = 0.87
Mutate to T: LLR = 0.87
Mutate to D: LLR = 0.72
Mutate to A: LLR = 0.64 Position 126 (Original: X):
Mutate to V: LLR = 5.51
Mutate to I: LLR = 5.07
Mutate to L: LLR = 4.74
Mutate to A: LLR = 4.02
Mutate to T: LLR = 3.96
Mutate to F: LLR = 3.36
Mutate to R: LLR = 3.28
Mutate to G: LLR = 3.26
Mutate to E: LLR = 3.18
Mutate to S: LLR = 3.08
Mutate to P: LLR = 3.07
Mutate to Y: LLR = 3.06
Mutate to C: LLR = 2.88
Mutate to M: LLR = 2.85
Mutate to K: LLR = 2.72
Mutate to D: LLR = 2.59
Mutate to W: LLR = 2.55
Mutate to Q: LLR = 2.46
Mutate to H: LLR = 2.39
Mutate to N: LLR = 1.89 Position 127 (Original: S):
Mutate to G: LLR = 1.53
Mutate to A: LLR = 1.52
Mutate to L: LLR = 1.44
Mutate to V: LLR = 1.37
Mutate to I: LLR = 1.23
Mutate to Y: LLR = 0.84
Mutate to F: LLR = 0.69
Mutate to T: LLR = 0.51 Position 128 (Original: X):
Mutate to V: LLR = 6.03
Mutate to A: LLR = 5.56
Mutate to L: LLR = 5.32
Mutate to I: LLR = 5.28
Mutate to T: LLR = 4.73
Mutate to G: LLR = 4.70
Mutate to F: LLR = 4.55
Mutate to S: LLR = 4.33
Mutate to M: LLR = 4.08
Mutate to Y: LLR = 3.94
Mutate to C: LLR = 3.80
Mutate to R: LLR = 2.84
Mutate to E: LLR = 2.66
Mutate to H: LLR = 2.64
Mutate to Q: LLR = 2.62
Mutate to W: LLR = 2.53
Mutate to N: LLR = 2.39
Mutate to D: LLR = 2.05
Mutate to P: LLR = 1.81
Mutate to K: LLR = 1.69 Position 129 (Original: F):
Mutate to L: LLR = 1.23
Mutate to I: LLR = 1.05
Mutate to V: LLR = 0.93 Position 130 (Original: T):
Mutate to L: LLR = 1.51
Mutate to I: LLR = 1.10
Mutate to V: LLR = 1.04 Position 131 (Original: X):
Mutate to V: LLR = 4.62
Mutate to I: LLR = 4.53
Mutate to L: LLR = 4.41
Mutate to A: LLR = 4.02
Mutate to G: LLR = 3.53
Mutate to F: LLR = 3.40
Mutate to S: LLR = 3.16
Mutate to M: LLR = 3.09
Mutate to T: LLR = 2.82
Mutate to C: LLR = 2.71
Mutate to Y: LLR = 1.81
Mutate to D: LLR = 1.67
Mutate to E: LLR = 1.65
Mutate to N: LLR = 1.64
Mutate to W: LLR = 1.44
Mutate to H: LLR = 1.37
Mutate to Q: LLR = 1.36
Mutate to R: LLR = 1.35
Mutate to P: LLR = 0.87 Position 137 (Original: V):
Mutate to R: LLR = 1.52
Mutate to G: LLR = 1.38
Mutate to S: LLR = 0.79
Mutate to A: LLR = 0.66
Mutate to D: LLR = 0.63 Position 138 (Original: I):
Mutate to A: LLR = 0.73
Mutate to S: LLR = 0.66
Mutate to L: LLR = 0.53 Position 139 (Original: D):
Mutate to F: LLR = 2.18
Mutate to W: LLR = 1.91
Mutate to P: LLR = 1.48
Mutate to L: LLR = 1.48
Mutate to Y: LLR = 1.07
Mutate to A: LLR = 0.53 Position 142 (Original: ):
Mutate to D: LLR = 0.74
Mutate to A: LLR = 0.69
Mutate to E: LLR = 0.59 Position 149 (Original: A):
Mutate to L: LLR = 1.31
Mutate to I: LLR = 0.67
Mutate to V: LLR = 0.53 Position 152 (Original: A):
Mutate to L: LLR = 1.36
Mutate to A: LLR = 1.14
Mutate to V: LLR = 0.83
Mutate to I: LLR = 0.71 Position 155 (Original: G):
Mutate to L: LLR = 1.54
Mutate to A: LLR = 1.41
Mutate to V: LLR = 0.68
Mutate to I: LLR = 0.65
Mutate to E: LLR = 0.63
Mutate to R: LLR = 0.61 Position 157 (Original: I):
Mutate to L: LLR = 2.75
Mutate to A: LLR = 2.72
Mutate to I: LLR = 1.95
Mutate to V: LLR = 1.88
Mutate to E: LLR = 1.73
Mutate to G: LLR = 1.66
Mutate to S: LLR = 1.64
Mutate to F: LLR = 1.55
Mutate to T: LLR = 1.35
Mutate to R: LLR = 1.11
Mutate to M: LLR = 1.03
Mutate to Q: LLR = 0.90
Mutate to D: LLR = 0.67
Mutate to K: LLR = 0.57 Position 161 (Original: I):
Mutate to E: LLR = 1.09
Mutate to A: LLR = 1.02
Mutate to D: LLR = 0.97 Position 162 (Original: S):
Mutate to A: LLR = 3.09
Mutate to E: LLR = 2.57
Mutate to R: LLR = 2.27
Mutate to S: LLR = 2.09
Mutate to T: LLR = 1.97
Mutate to D: LLR = 1.89
Mutate to K: LLR = 1.80
Mutate to Q: LLR = 1.79
Mutate to G: LLR = 1.70
Mutate to L: LLR = 1.47
Mutate to P: LLR = 1.05
Mutate to V: LLR = 1.02
Mutate to N: LLR = 0.98
Mutate to H: LLR = 0.90 Position 174 (Original: A):
Mutate to A: LLR = 3.50
Mutate to R: LLR = 3.12
Mutate to S: LLR = 3.01
Mutate to T: LLR = 2.99
Mutate to L: LLR = 2.88
Mutate to K: LLR = 2.82
Mutate to G: LLR = 2.71
Mutate to E: LLR = 2.64
Mutate to D: LLR = 2.51
Mutate to V: LLR = 2.51
Mutate to Q: LLR = 2.31
Mutate to N: LLR = 2.19
Mutate to I: LLR = 2.11
Mutate to P: LLR = 1.95
Mutate to H: LLR = 1.79
Mutate to M: LLR = 1.36
Mutate to F: LLR = 1.32
Mutate to Y: LLR = 0.83 Position 178 (Original: P):
Mutate to A: LLR = 1.11
Mutate to R: LLR = 0.96
Mutate to Q: LLR = 0.52 Position 181 (Original: A):
Mutate to E: LLR = 2.23
Mutate to D: LLR = 1.54
Mutate to R: LLR = 1.26
Mutate to Q: LLR = 0.98
Mutate to A: LLR = 0.85
Mutate to K: LLR = 0.67 Position 182 (Original: T):
Mutate to V: LLR = 4.57
Mutate to I: LLR = 3.69
Mutate to L: LLR = 3.16
Mutate to F: LLR = 2.15
Mutate to A: LLR = 2.15
Mutate to C: LLR = 1.91
Mutate to T: LLR = 1.13
Mutate to M: LLR = 0.77 Position 185 (Original: R):
Mutate to L: LLR = 2.84
Mutate to A: LLR = 1.98
Mutate to G: LLR = 1.76
Mutate to M: LLR = 1.67
Mutate to R: LLR = 1.53
Mutate to Q: LLR = 1.14
Mutate to S: LLR = 1.00
Mutate to E: LLR = 0.96
Mutate to H: LLR = 0.74
Mutate to T: LLR = 0.71
Mutate to D: LLR = 0.67
Mutate to F: LLR = 0.64
Mutate to K: LLR = 0.57
Mutate to Y: LLR = 0.56 Position 188 (Original: A):
Mutate to E: LLR = 3.31
Mutate to A: LLR = 2.80
Mutate to D: LLR = 2.68
Mutate to S: LLR = 2.50
Mutate to Q: LLR = 2.36
Mutate to T: LLR = 1.84
Mutate to N: LLR = 1.65
Mutate to G: LLR = 1.42
Mutate to L: LLR = 1.39
Mutate to R: LLR = 1.37
Mutate to K: LLR = 1.36
Mutate to H: LLR = 0.71 Position 192 (Original: T):
Mutate to V: LLR = 7.46
Mutate to N: LLR = 7.19
Mutate to Q: LLR = 6.97
Mutate to I: LLR = 6.92
Mutate to A: LLR = 6.88
Mutate to L: LLR = 6.77
Mutate to T: LLR = 6.67
Mutate to D: LLR = 6.52
Mutate to E: LLR = 6.46
Mutate to S: LLR = 6.35
Mutate to P: LLR = 6.23
Mutate to Y: LLR = 6.07
Mutate to M: LLR = 5.65
Mutate to G: LLR = 5.41
Mutate to R: LLR = 5.30
Mutate to H: LLR = 5.06
Mutate to F: LLR = 4.99
Mutate to K: LLR = 4.49
Mutate to W: LLR = 3.79
Mutate to C: LLR = 3.53 Position 198 (Original: D):
Mutate to A: LLR = 1.24
Mutate to E: LLR = 0.90
Mutate to L: LLR = 0.81
Mutate to I: LLR = 0.68 Position 199 (Original: V):
Mutate to L: LLR = 3.77
Mutate to M: LLR = 1.63
Mutate to V: LLR = 1.14
Mutate to I: LLR = 0.95
Mutate to F: LLR = 0.67
Mutate to T: LLR = 0.61 Position 202 (Original: V):
Mutate to S: LLR = 1.98
Mutate to P: LLR = 1.27
Mutate to A: LLR = 1.20 Position 203 (Original: K):
Mutate to V: LLR = 2.31
Mutate to P: LLR = 1.82
Mutate to I: LLR = 1.59
Mutate to L: LLR = 1.38
Mutate to E: LLR = 1.20
Mutate to A: LLR = 1.18
Mutate to R: LLR = 0.76
Mutate to T: LLR = 0.67 Position 208 (Original: R):
Mutate to T: LLR = 1.82
Mutate to N: LLR = 1.48
Mutate to S: LLR = 1.02 Position 209 (Original: V):
Mutate to H: LLR = 3.42
Mutate to Y: LLR = 2.66
Mutate to R: LLR = 1.62
Mutate to Q: LLR = 0.73
Mutate to L: LLR = 0.59 Position 212 (Original: R):
Mutate to N: LLR = 2.55
Mutate to S: LLR = 1.41
Mutate to R: LLR = 0.98
Mutate to A: LLR = 0.88 Position 214 (Original: E):
Mutate to M: LLR = 8.36
Mutate to Y: LLR = 8.16
Mutate to L: LLR = 8.13
Mutate to R: LLR = 7.78
Mutate to F: LLR = 7.65
Mutate to E: LLR = 7.36
Mutate to A: LLR = 7.24
Mutate to Q: LLR = 7.22
Mutate to K: LLR = 7.17
Mutate to I: LLR = 7.12
Mutate to V: LLR = 6.98
Mutate to S: LLR = 6.58
Mutate to C: LLR = 6.40
Mutate to T: LLR = 6.04
Mutate to W: LLR = 5.67
Mutate to H: LLR = 5.62
Mutate to G: LLR = 5.38
Mutate to N: LLR = 4.57
Mutate to D: LLR = 3.21
Mutate to P: LLR = 1.85 Position 224 (Original: K):
Mutate to E: LLR = 2.96
Mutate to Q: LLR = 2.18
Mutate to D: LLR = 1.11
Mutate to A: LLR = 0.56

Total hotspots found: 75”

These results seemed a bit too excessive so I changed the code around a little bit and got this

Mutation Hotspots Identified (positions with at least 3 mutations having LLR > 5):

Position 20 (Original: I):

Mutate to E: LLR = 5.70
Mutate to A: LLR = 5.51
Mutate to D: LLR = 5.31 Position 128 (Original: X):
Mutate to V: LLR = 6.03
Mutate to A: LLR = 5.56
Mutate to L: LLR = 5.32
Mutate to I: LLR = 5.28 Position 192 (Original: T):
Mutate to V: LLR = 7.46
Mutate to N: LLR = 7.19
Mutate to Q: LLR = 6.97
Mutate to I: LLR = 6.92
Mutate to A: LLR = 6.88
Mutate to L: LLR = 6.77
Mutate to T: LLR = 6.67
Mutate to D: LLR = 6.52
Mutate to E: LLR = 6.46
Mutate to S: LLR = 6.35
Mutate to P: LLR = 6.23
Mutate to Y: LLR = 6.07
Mutate to M: LLR = 5.65
Mutate to G: LLR = 5.41
Mutate to R: LLR = 5.30
Mutate to H: LLR = 5.06 Position 214 (Original: E):
Mutate to M: LLR = 8.36
Mutate to Y: LLR = 8.16
Mutate to L: LLR = 8.13
Mutate to R: LLR = 7.78
Mutate to F: LLR = 7.65
Mutate to E: LLR = 7.36
Mutate to A: LLR = 7.24
Mutate to Q: LLR = 7.22
Mutate to K: LLR = 7.17
Mutate to I: LLR = 7.12
Mutate to V: LLR = 6.98
Mutate to S: LLR = 6.58
Mutate to C: LLR = 6.40
Mutate to T: LLR = 6.04
Mutate to W: LLR = 5.67
Mutate to H: LLR = 5.62
Mutate to G: LLR = 5.38

Total hotspots found: 4

One thing I noticed whilst analysing the heatmap, the x spots around 16, 122 to 126, 189 and 211 appear brighter. That means that these are the areas where the protein’s sequence might be quite adaptable, making them interesting for further investigation into their role and potential for modification.

New sequence for folding: MNFLDDVEKLSKVKGDDETFKKGLEEIAKKHGFSGWNFVYDNNGHLKVWSNLDPALLKEYFEKNWYKVDPVIKKAREEEKLFTWSWDEEWPKLTEEEKALGEEYKKYGVKSGITIPIKAENGTRAYFTFYSKKDKIELKEPIDDEKAKKVTKLLYKKIVDNKWTPTGHTPMSLNEKERAYLKLRSKGYSQREIAEKLGVAQSTVSRTIRKAMEKFGVKTIEELIKIAKEMGLI

What it looks like

Part D. Group Brainstorm on Bacteriophage Engineering

*Claude AI prompt: Can you please analyse the results of UniProtKB LuxR search ** Gemini built in to colab AI prompt “identify mutation hotspots based on the compiled data in the heatmap”