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SolutionSet2 - BMB100B Winter 2009 Rubin Solution Set#2 Due...

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BMB100B Winter 2009 Rubin Solution Set #2 Due Tuesday, January 20 th 10:00 A.M. 1) Go to the Protein Data Bank and download the structure of the Fos-Jun-DNA complex (PDB ID: 1FOS). The proteins Fos and Jun comprise a transcription factor that is deregulated in several cancers. This structure of the heterodimer is solved in the presence of DNA, but here we are interested in the protein heterodimer itself. Note also that two copies of the ternary complex appear in the PDB file because of the crystallographic packing. a) What structural motif is represented by the heterodimer? coiled-coil b) Draw a helical wheel diagram for ~6 turns of each helix. Begin with K176 of Fos (Chain E or G) and of N300 Jun (Chain F or H) and include the remaining sequence to the C- terminus for each. Placing K176 and N300 of Fos and Jun respectively into position A of the helical wheel one gets the following picture: Note: If you start with A178 of Fos and E302 of Jun, these would have to go into position C based on the structure. c) Based on your diagram, explain why a hypothetical structure of a Fos or Jun homodimer would be less stable than the heterodimer. The favorable electrostatic contribution is only possible with a heterodimer. For example, if you look at the G position in Fos and the E position in Jun, there are negative and positive charges that can interact. A Fos-Fos or Jun-Jun homodimer would not have favorable electrostatics at these positions. 2) Suppose you are purifying the E. coli maltose-binding protein (MBP) from bacteria cell lysates (i.e. a mixture of all of the soluble proteins containing the bacteria).
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BMB100B Winter 2009 Rubin a) What type of ion exchange chromatography would you use and at what buffer pH would you carry out your purification? You may find typing “isoelectric point calculator” into Google helpful… The amino acid sequence for MBP can be copied from the link off the PDB:1ANF entry (click on FASTA Sequence on left). You could also have found the sequence using other protein databases (Swiss Prot, NCBI, etc.). Using an on-line isoelectric point calculator (e.g. http://www.embl-heidelberg.de/cgi/pi-wrapper.pl), the PI=5.03. If pH > PI, the protein is negatively charged. Thus, anion exchange in a buffer of pH=7 would be suitable.
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