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Key Links: You can read more about the final project in the Final Project Page.
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Bacteriophage MS2 is a single stranded RNA virus whose genome only encodes 4 proteins -the maturation protein (A-protein), the lysis (L-Protein) protein, the coat protein (cp), and the replicase (rep) protein. Bacteriophages infect E-coli. Upon infection, the L-Protein forms pores in the E-coli cell membrane which eventually leads to breakdown of the membrane (Lysis). DnaJ is a chaperone protein in E-coli (chaperone proteins are proteins that assist during protein folding). It is thought to be involved in the lysis mechanism. In this homework, we will explore if computational models we learnt about in the last class are useful for designing variants/mutants of the lysis protein sequence. We will study the effects of L-protein mutants on the bacteriophage infectivity.
Our goal for this part of the homework is to create mutants of L-protein that affect its lysis activity and/or its interaction with DNAj. Making a mutation for L-protein without a way to computationally predict what happens to lysis or its interaction with DNAj is hard. So we are going to try various hypotheses on how to use the models from last week and also try a few other tools. These mutants will be tested in the lab.
Lysis Protein Sequence (UniProtKB ID: https://www.uniprot.org/uniprotkb/P03609/entry**)**
METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT
Lysis protein contains a soluble N-terminal domain followed by a transmembrane protein (blue/last 35 residues). Transmembrane protein affects the lysis activity. The soluble domain (green) is the domain responsible for interaction with DNAj.
DNAj sequence (UniProtKB ID: https://www.uniprot.org/uniprotkb/P03609/entry**)**
MAKQDYYEILGVSKTAEEREIRKAYKRLAMKYHPDRNQGDKEAEAKFKEIKEAYEVLTDSQKRAAYDQYGHAAFEQGGMGGGGFGGGADFSDIFGDVFGDIFGGGRGRQRAARGADLRYNMELTLEEAVRGVTKEIRIPTLEECDVCHGSGAKPGTQPQTCPTCHGSGQVQMRQGFFAVQQTCPHCQGRGTLIKDPCNKCHGHGRVERSKTLSVKIPAGVDTGDRIRLAGEGEAGEHGAPAGDLYVQVQVKQHPIFEREGNNLYCEVPINFAMAALGGEIEVPTLDGRVKLKVPGETQTGKLFRMRGKGVKSVRGGAQGDLLCRVVVETPVGLNERQKQLLQELQESFGGPTGEHNSPRSKSFFDGVKKFFDDLTR
Additional Information
Using the mutational analysis of lysis protein from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775895/ Here is a list of some mutations and their effect on lysis https://docs.google.com/spreadsheets/d/11WzDDNkQDEiqbUSGV0ZCqITGctyNFpD7xnPlhsj2BhE/edit#gid=0 [Three columns highlighted ! Please copy the sheet before any edits).
In the last class, we used pBLAST to find evolutionary sequences from other organisms and visualized it using clustalomega. BLAST results for L-protein are here - https://drive.google.com/drive/folders/1eQeuwL9WiO16bw6Lb8z-TVpbWIoAF4EH?usp=share_link . You can upload it to clustalomega - https://www.ebi.ac.uk/jdispatcher/msa/clustalo to get the alignments. Usually a good idea to not make mutations in the conserved sites (the amino acid positions where you see no changes in the alignments)
Additional Information about the sequence is linked here - https://www.notion.so/howtogrowalmostanything/L-Protein-Sequence-Information-7a29684affaa4e3b9adf070cbb7c9908?pvs=4
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You have the choice to pick between Option 1 and Option 2.
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