Group Final Project

L-Protein Engineering 

The idea is to transform L-Protein into a self-stabilizing, autonomously localized structure, eliminating the need for DNAJ assistance. This will improve the stability of L-Protein.

STEP 1

Gather Information about Lysis Protein /Sequence /DNAj Sequence / Conserved sites if any Known mutational effects from research

Lysis Protein Sequence (UniProtKB ID: https://www.uniprot.org/uniprotkb/P03609/entry)

METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT

DnaJ sequence (UniProtKB ID: https://www.uniprot.org/uniprotkb/P03609/entry)

MAKQDYYEILGVSKTAEEREIRKAYKRLAMKYHPDRNQGDKEAEAKFKEIKEAYEVLTDSQKRAAYDQYGHAAFEQGGMGGGGFGGGADFSDIFGDVFGDIFGGGRGRQRAARGADLRYNMELTLEEAVRGVTKEIRIPTLEECDVCHGSGAKPGTQPQTCPTCHGSGQVQMRQGFFAVQQTCPHCQGRGTLIKDPCNKCHGHGRVERSKTLSVKIPAGVDTGDRIRLAGEGEAGEHGAPAGDLYVQVQVKQHPIFEREGNNLYCEVPINFAMAALGGEIEVPTLDGRVKLKVPGETQTGKLFRMRGKGVKSVRGGAQGDLLCRVVVETPVGLNERQKQLLQELQESFGGPTGEHNSPRSKSFFDGVKKFFDDLTR

Known Mutational Effect

LS dipeptide: Positions 44 and 45 (Leucine-Serine) in the MS2 L protein are crucial. Domain division: The L protein is divided into four domains. Domain 1 (N-terminus), while positively charged and important, is not essential for the cleavage function itself (primarily responsible for binding to the host chaperone protein DnaJ); while Domains 2 to 4 (C-terminal half), containing the LS motif, are the key components for performing the cleavage function.

Therefore, the design mainly focuses on the Domain 1 region and replaces the water-soluble amino acids in it to improve its hydrophobicity and make it stable to generate spontaneous folding.

STEP 2

Select an approach to make sequence variants

Plan 1: METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLLVLIFLAIFLSLFTNQLLLSLLEAVIRTVTTLQQLLT

Design Actions: According to the LLR score, the K mutation at position 50, changing to L, yields a score of 2.56. Replacing with L (leucine) significantly enhances the hydrophobic anchoring force at the Domain 2/4 junction. Changing the Y at position 39 to L, located in Domain 1, yields a score of 2.24. Y contains a polar hydroxyl group; replacing it with L makes the transmembrane helix purer and more stable.

Plan 1.5: METRFPQQSQQTPASTNRRRPFKHEDYPRRRQQRSSTLLVLIFLAIFLSLFTNQLLLSLLEAVIRTVTTLQQLLT

Design Actions: According to the LLR, the C mutation, changing to R, yields a score of 2.39.

Principle: Increasing positive charge enhances the protein’s autonomous attraction to the negatively charged cell membrane, thereby reducing dependence on DNAJ escort.

Plan 2: METRFPQQQQQTPASTNRRRPFKHEDYPRRRQQRSSTLLVLIFLAIFLSLFTNQLLLSLLEAVIRTVTTLQQLLT

Design Action: According to LLR, mutating S to Q results in a score as high as 2.39. Principle: Increasing the rigidity of Domain 1 allows it to fold into a helical state.