Protein Structure Worksheet Biol 215 Name___________________________________________ Cystic fibrosis is a hereditary disease affecting the sweat and mucus

glands of afflicted individuals, resulting in the production of thick, viscous mucus. The gene responsible for this disease, Cystic fibrosis transmembrane conductance regulator (CTFR), has been identified and found to be recessive. The CTFR gene codes for a transmembranespanning chloride ion-channel protein. Although the exact molecular mechanisms involved in the development of cystic fibrosis symptoms are not known, defects in the CTFR prevent the channel from functioning properly, leading to a blockage of the movement of salt and water into and out of cells. 1) If this is a genetic disease, then why is the function of an ion-channel protein affected? The defected genes code for the incorrect proteins, therefore causing the ion-channel protein affected.

2) A partial DNA coding sequence for the CTFR gene is shown below. If the sequence were shortened by removing the highlighted nucleotide, what would be the expected result when compared to the original polypeptide? 5-ATGCAGAGGTCGCCT-3 a) The sequence of amino acids would be unchanged. b) The polypeptide sequence will be shortened by one amino acid. c) The polypeptide sequence would shortened by several amino acids. d) There would be a different sequence of amino acids after the deletion. e) No product would be produced. Answer: (D)

Shown below is a diagram of the CTFR protein within an endothelial cell. Note: keep in mind that most cells in our bodies are bathed in fluids; i.e. the extracellular space is also aqueous.

MSD = Membrane-spanning domain NBD = Nucleotide-binding domain R = Regulatory domain

3) Is the NBD1 closer to the amino-terminus or the carboxy-terminus? NBD1 is closer to the carboxyl-terminus.

4) Of the four classes of amino acids (nonpolar, polar uncharged, acidic, basic), which class would you expect to find in the MSDs? Explain. Polar, uncharged. Would you expect the loops connecting the membrane-spanning segments (i.e. the short black lines between the yellow bars) to have the same characteristics? Explain. Yes. They need to connect continuously.

5) Of the four classes of amino acids, which class would you expect to find in the cytoplasmic domains interacting with the cytoplasm? Explain. Polar. It can then interact in aqueous solution.

6) There are currently 1800+ known mutations in the CFTR gene. One particularly common CF mutation, F508, involves the deletion of three specific base-pairs found in the first NBD. Shown below are portions of the coding strands from the CFTR gene. Translate the wild-type and mutant sequences. What change, if any, occurs in the mutant polypeptide sequence? Wild-type CFTR: 5-ATC ATC TTT GGT GTT-3 F508 CFTR: 5-ATC ATT GGT GTT-3 A Pro becomes a Lys which is incorrect.

7) The F508 mutation is known to affect the targeting of the CFTR protein. The mutant protein never makes it to the Golgi. What might cause the protein to not be properly targeted? Incorrect genes code for incorrect receptors, therefore the proteins are not properly targeted. In terms of proper subcellular targeting, do you think this mutation will cause a change in phenotype? Explain. Yes, the alteration in genes will cause a different phenotype.

8) Diagram the steps involved in targeting the CFTR protein to the plasma membrane. The proteins go through the rough ER, then to the lipid bilayer, then degrades.

9) The CFTR protein is 1480 amino acids long. Identify the mutation in the following mRNA transcripts. Then translate the segments and predict the changes (if any) in the resultant mutant peptide. a. At amino acid (aa) 1400 Normal: 5-GAG GAG-3 Mutant 5-AAG GAG-3 Glu, Glu -> Lys, Glu b. At aa 327: Normal: 5-CUU CUA GUU-3 Mutant 5-CUU UAG UUU-3 Leu, Leu, Val-> Leu, stop c. At aa 742: Normal: 5-AGU AGC-3 Mutant 5-ACU AGU-3 Ser, Ser-> Thr, Ser

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