Problem Set 3 BIBC 100 Spring 2008 1. For each of the following amino acid substitutions, indicate if they would be apt to cause a change or disruption of protein structure and explain your answer. Val to Leu Leu to Lys Asp to Glu Ala to Tyr 2. Which of the following peptides do you think would form a more stable alpha helix and why? #1. A – V – R – M – W – V – E – L – S #2. R – K – R – W – Q – K – R – M - H – W3. You have just completed a mass spectrometry experiment and you have successfully determined the molecular mass of your 100 amino acid protein. For the experiment, you treated your protein with the enzyme trypsin, which cleaves after all Lys and Arg residues. Your protein has Lys at positions 25, 65 and 82 and Arg at positions 14, and 58. a. How many amino acids would you have in each of the resulting proteolytic fragments? b. Estimate the molecular weight of each of these fragments. (Remember that the mass spec technique would give you exact masses of each of these fragments) c. What information do you gain by cleaving your peptide with trypsin and running this experiment? 4. The Greek letter theta (Θ) is used to label the Y-axis in all of the oxygen binding plots or ligand binding plots presented in class. Explain in a sentence or phrase what theta represents.5. Go to NCBI and search (use the drop down menu next to “Search”) the Protein database for Myoglobin. How many sequences do you retrieve? This is too many sequences to look through, especially if you are only wanting the human Myoglobin sequence! Click on the tab marked “RefSeq” (reference sequences). The first 3 sequences listed here are all human Myoglobins. What similarities/ differences can you see between these three myoglobin sequences? Open the record for the first human myoglobin entry. Notice that you bring up a screen that gives a lot of information about this myoglobin entry. However, you only want the protein sequence of myoglobin to BLAST (listed at the very bottom of this record). At the top of the screen, change the pull down menu marked “Display” from GenPept to FASTA. Now, the record has been simplified to just the protein sequence with a comment line (starts with “>”). Copy this sequence, and go to the NCBI BLAST page. Notice the option “BLAST assembled genomes”. You want to determine how many protein sequences are similar to your human myoglobin sequence in the human genome, so select the “Human” link. Paste your sequence (notice it is in FASTA format) into the box. For the “Database” drop down menu, choose “RefSeq protein” – this is a compilation of all known human proteins. For the “Program” drop down menu, choose “BLASTP”, because you are comparing your protein sequence to all human protein sequences. Notice the “Expect” menu…we will change it in a second. Hit the “Begin Search” button. On the next page, you will have to hit “View Report” to see your results…it may take a little time for your BLAST results to be computed. How many proteins (different from your myoglobin) are retrieved as matches?How similar is the most similar match? How similar is the least similar match? Now, repeat this search, and change the “Expect” menu to “10”. Does this make your search more or less stringent? Beyond what Expect value do you think the matches become
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