FST 201Final Exam

Due at 4:00 pm, Tuesday, December 6, in 3208 RMI South (or my mailbox in the main office)

1) The following table lists some well-known chemical modification reagents. Indicate the most common sidechain(s) that ismodified by each reagent, and a possible outcome of making the modification (i.e., why you would want to do it).

Reagent / AA residue / Effect on structure, function, etc.
N-Ethyl maleimide
Diethyl pyrocarbonare
Succinic anhydride
Acetic anhydride
Iodoacetamide
Acetaldehyde + Na(CN)BH4
TPCK

2) You have detected an enzyme activity that seems to break down starch in an extract of a bean from a leguminous bush You think that the enzyme might be commercially useful, so you want to clone its gene for over-expression in a GRAS host. You have a rapid screening method in which you can detect the presence of the enzyme (e.g., a drop of the extract on a starch gel, followed by detection of new reducing ends), a method of transformation and a transposon from a similar species. You transform You find a sample that does not contain the enzyme, which you presume was created when the transposon inserted itself into the gene encoding the enzyme. Of course, none of this matters for answering the question.

a. Before you go to the trouble of sequencing the entire gene, you want to know if the insertion ( and the gene) is where you think it is. So, you use a primer that is homologous to the transposon, and sequence a little piece of the adjacent region. You obtain the following sequence:

gctggttggactgccgatgctgatggaactactacatgaacgacgtactcgccgcgctcgcgcgtgaggctggctgggtcgtcgacgccgacggcaccacctaccgccagtgcccccctccttctcacatggggtcat

From this information alone, does it look as though the transposon is inserted in the gene you want to sequence? What makes you think so (or not)?

b. Based on your answer to part a, you sequence the DNA on both flanks of the transposon and obtain the following nucleotide sequence:

gtgaagaagcgagaagaggaagttgagtgagaaccgtgaaaattaaattgtgaagatgaagagcgtatctgattatttatcaacccaagatctcgaccctcagagcgaccacagctccgattacttagctcacccccaaccccaccctcagccccgccgtcctcggggtttcgcggcggcagcggcggcggccaccaccaattccgccggcaaggggaagaaggagagggagaaggagaaggagcgcaccaagctccgcgagcgacaccgccgagccatcaccagccgcatgctcgccggactccgccgctggttggactgccgatgctgatggaactactacatgaacgacgtactcgccgcgctcgcgcgtgaggctggctgggtcgtcgacgccgacggcaccacctaccgccagtgcccccctccttctcacatggggtcatttgcggctagatcagttgaaagtcaactctctggtggttctctaaggaattgctctgttaaggaaacgactgagaatcagacatctgtgcttagaattgatgaatgcttgtcgcctgcatccattgattctgttgtaattgcagaaagggactcgaagactgagaaatatacaaatgctagccccatcaatacagttgactgcttggaggctgatcagcttatgcaagatattcattctggggtgcatgaattttgccagttgatggatcctgaaggcattaggcaggagctaatccatattaagtccttaaatattgacggtgttttgattgatcctgaaggcataaagcaggagctcatccatatcaagtctttaaatgtagatggtgttgttgtggattgttggtggggcattattgaaggctggagctcacagaaatatgtgtggtctggttatagggagctttttaacattattagagaattcaaactgaagttacaggttgttatggcatttcatgaatgtggagggaatgattctagtgatgcattaatttccctcccacaatgggttttggatattggaaaagacaaccaagatatattcttcacagatcgtgaaggacggaggaatactgaatgcctttcttgggggattgacaaagaacgagttctcaaaggcagaactggaattgaggtctattttgatatgatgagaagctttcggacagagtttgatgacctgtttgcagaagtctcagaaagaatgggatggaggtatcctggtatcggtgagtttcagtgctatgataaatacctgcagcatagtctgcgcagagcagccaaattacgtggtcactctttctgggctagaggacctgataatgctggacattacaattctatgccacatgaaactggattcttttgtgagcgaggtgattatgacaactattatggacgcttcttcttacattggtactcccagacattaatagaccatgcagataatgttttgtctcttgcaacacttgctttcgaggaaacaaagataactgtcaaggttcccgctgtatactggtggtataagactcctagtcatgctgcagagttgacagcaggatatcacaatccaacaaatcaggatggatattctcctgtgtttgaggttctgagaaaacatgctgtcaccatgaaatttgtctgcttaggatttcatctttccagccaggaagctaatgaatcattgattgatccagagggtttgagttggcaggtgctaaactcagcttgggatcgcgggttgatggctgctggagagaatgcactcctttgctatgatagagaagggtacaagaaattggttgagattgcgaagcccagaaatgaccccgatcgccgacacttctcattctttgtttatcagcaaccatctttgctgcagacaaatgtttgctggtccgaactggatttctttgtcaaatgcatgcatggtgagatgacagatctataatggcggcaaaattcacgtcttaactgtaggccatttcatgggggcgtaatgaccttgtggcagtacaatgccatttgtaacaatatcatctatatataatattgacagccttcaagaggtggccagcccccaagcagcaccgtaccatattagatcagtgctttttgcaagcttggtaatgccagtctaagttctacggcattccaacttctgatctatctaaccgataaaactggcagttatgaaatgtgattatggatggccaaatttcatgaagaaatttgttggcaataataaaaagcaaatatatgtct

What is the amino acid sequence of the protein that this gene encodes? (Assume that it starts with the first Met residue.)

c. In what ways could the actual sequence of the protein differ from the deduced sequence?

d. Is this likely to be a transmembrane protein? Provide evidence for your answer.

e. Obtain at least two secondary structure predictions for this protein. Comment on their agreement or lack thereof.

f. What is the closest relative of this protein? (A species name is not a relative – the questing asks what enzymes are related to this one)

3. What would you expect to be the effect of the following treatments on the functional properties of a “typical” protein (e.g., a whey protein)? You can write “increase”, “decrease”, “no effect”, or give a brief expression of your thoughts.

Treatment
/effect on / solubility / foaming / emulsification / gel formation
Partial denaturation by heating
Slight hydrolysis by trypsin
Extensive hydrolysis by trypsin
Addition of 1M NaCl
Addition of nonionic detergent
Addition of reducing agents

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