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Using bioinformatics tools to identify conserved sequences and frame a research question

Using bioinformatics tools to identify conserved sequences and frame a research question. Cynthia Brame ASM/JGI Bioinformatics Institute 2012. CK1s are part of the eukaryotic protein kinase superfamily. Manning et al. Science 298: 1912-1918 (2002). Learning objectives. Big goal:

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Using bioinformatics tools to identify conserved sequences and frame a research question

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  1. Using bioinformatics tools to identify conserved sequences and frame a research question Cynthia Brame ASM/JGI Bioinformatics Institute 2012

  2. CK1s are part of the eukaryotic protein kinase superfamily Manning et al. Science 298: 1912-1918 (2002).

  3. Learning objectives • Big goal: • Students will understand the importance of conserved sequences in protein function. • Smaller goals: • Students will use bioinformatics tools • to identify a conserved domain within a protein. • to identify and display conserved sequences within a protein. • to map conserved residues on to a 3D protein model to form hypotheses about function.

  4. Starting point: Amino acid sequence for CKI from Schizosaccharomycespombe • Search Pfam with the sequence. • Protein kinase domain. • Look at the sequence alignment. • Capital letters vs. lower-case letters • Show them a model of a protein kinase and point out what the most highly conserved amino acids do.

  5. What does the model indicate is highly conserved that is not conserved in our kinase? • Non-conservative substitutions at conserved residues • Insertions

  6. Are these just mutations in this kinase, or are they found more broadly? • Introduce the idea of superfamilies, families, and subfamilies. Manning et al. Science 298: 1912-1918 (2002).

  7. Are these sites conserved in the CK1 family? • Have students search the NCBI protein database for other CK1s. • Get representatives of all CK1 subfamilies (alpha, beta, gamma, delta, preferably from multiple species). • Get Yck2 from S. cerevisiae(for web lab work).

  8. Are these sites conserved in the CK1 family? • Have students perform multiple sequence alignment. • http://www.phylogeny.fr/ • Export as a fasta file. • Have students create a visual depiction of their results with Weblogo. • http://weblogo.berkeley.edu/logo.cgi

  9. Are these sites conserved in the CK1 family?

  10. Where are these sites found on the protein structure? • Map multiple sequence alignment onto 1CSN, a crystal structure-based model of CKI from S. pombe. • http://consurf.tau.ac.il/

  11. Based on location, have students develop hypotheses about function of these residues. • Ideally, they will suggest hypotheses that can be tested by mutating the residues. • E.g., T166 is highly conserved in CK1s; students might suggest is regulatory phosphorylation site. Could do two mutations: T166E and T166A to test. • The mutation can also be modeled using VMD to see the effect on protein structure.

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