1) Cystic fibrosis is a serious genetic disease, with disorders of the glandular function of several organs, including the lungs, gastrointestinal tract and pancreas. The disease leads to the formation of an abnormally tough mucus that closes glands and discharge passages, especially in the lungs, sinuses, pancreas and male genitals. The disease therefore typically causes respiratory problems, but also affects (affects) fertility, digestion and other bodily functions. Cystic fibrosis is caused by mutations in the transmembrane conductance regulator CFTR (CFTR), and is an autosomal recessive inherited disease.

a) What would be typical properties of the amino acids that are in the parts of the CFTR protein that traverse (pass through) the cell membrane? Explain why. How can this be used to predict (predict / calculate) whether a protein has transmembrane regions based on its amino acid sequence? (The answer must be at least 10 lines)

Transmembrane proteins are those proteins that span the entire lipid bilayer present in the cell membrane. They allow the easy transport of specific substances across the membrane. Since lipids are hydrophobic in nature, the transmembrane proteins are usually hydrophobic in the middle with hydrophilic regions at the ends. The hydrophobic regions will have amino acids that are non-polar in nature, while the hydrophilic regions will have predominantly polar amino acids. CFTR protein is an ion channel that moves molecules or atoms with an electrical charge from inside to outside the cell and vice versa. The CFTR protein contains 1,480 amino acids, the majority of which need to be hydrophobic. The amino acids tryptophan and tyrosine have aromatic side chains that can contribute to the hydrophobicity of the protein. The presence of non-polar amino acids with hydrophobic side chains includes alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan can be seen within the hydrophobic region of the protein (those that lie embedded within the lipid bilayer). Amino acids with polar but uncharged amino acids like serine, threonine, cysteine, asparagine, glutamine, and tyrosine will probably be found on the surface of the protein.

CFTR is a so-called ABC transporter protein that binds ATP intracellularly and transports chloride ions across the cell membrane

b) The CFTR protein has multiple transmembrane domains. Explain why proteins are often organized in domains. (The answer must be at least 10 lines)

The proteins are polypeptides formed by joining several amino acids using the peptide bond. The primary structure of protein then folds to form secondary structures like ï¡-helix, ï¢-sheets, etc. These secondary structures further fold to form tertiary structures. Every protein has a primary, secondary, and tertiary structure. A protein domain is a conserved part of a protein sequence or tertiary structure that plays a specific role, can evolve, function, and exist independently of the remaining protein chain. This organization into domains allows them to be arranged in different ways to create proteins with varying functions. Thus, domains are the functional units of the proteins. Since every domain can fold individually, the folding process in protein takes place in less time, thereby preventing any unwanted interaction between amino acid residues taking place. Domains also help in burying hydrophobic residues inside while allowing hydrophilic residues to remain on the surface. Proteins are allowed flexibility due to the domains.

A cell can have many different types of transmembrane proteins, with different functions

c) Why do cells need to have transmembrane proteins in order to maintain their existence? Describe examples of this. (The answer must be at least 15-20 lines)

Transmembrane proteins, as the name specifies are proteins that span the entire cell membrane. They act as a channel between the contents within the cell and those outside the cell. Since the cell membrane has a lipid bilayer, the transmembrane proteins have three domains; an extracellular domain which remains outside the cell, an intracellular domain, which remains completely within the cell, and the domain which remains within the lipid bilayer. These transmembrane proteins play an important role in communicating with the outside of the cell apart from working as transporter channels or carrier proteins. Porins are an example of a transmembrane protein that helps in creating pores or channels within the membrane. They are beta-barrel proteins (ï¢-sheets made of ï¢-strands linked together by ï¢ turns) which act as a pore/channel and allow molecules to diffuse through the membrane. Theses are found in bacteria as well as eukaryotes and a similar channel has been discovered in archaebacteria too. They help in transporting hydrophilic molecules of various sizes and charges, passively through the membrane. This is helpful in transporting wastes and toxins out of the cell and transporting nutrients and substrates within the cell. They can also regulate permeability and prevent lysis by limiting the amount of detergent entering the cell. Porins are of two types, general and selective. As the name specifies, the former has no substrate specificity but might show a preference for anions or cations. The latter is specific for chemical species. Porins have a threshold size of transportable molecules.

In the text below, parts of the CFTR mRNA sequence are listed (retrieved from the NCBI database; Homo sapiens CF transmembrane conductance regulator (CFTR), mRNA). Although mRNA actually contains Uracil, U, instead of Thymine, T (found in DNA), mRNA sequences in this database are nevertheless written with "DNA nomenclature":

1-GTAGTAGGTCTTTGGCATTAGGAGCTTGAGCCCAGACGGCCCTAGCAGGGACCCCAGCGC-60 61-CCGAGAGACCATGCAGAGGTCGCCTCTGGAAAAGGCCAGCGTTGTCTCCAAACTTTTTTT-120
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1501-TTCAGAGGGTAAAATTAAGCACAGTGGAAGAATTTCATTCTGTTCTCAGTTTTCCTGGAT-1560
1561-TATGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATGATGAATATAGATA-1620 1621-CAGAAGCGTCATCAAAGCATGCCAACTAGAAGAGGACATCTCCAAGTTTGCAGAGAAAGA-1680

The numbers on each side refer to the nucleotide number in the mRNA molecule.

d) Where (at what nucleotide number in the mRNA sequence) would you expect translation start? Justify the answer. (The answer must be at least 3 lines)

The translation starts with the AUG or methionine codon. Since here the mRNA sequence is written in DNA nomenclature, the sequence ATG on the coding strand will correspond with the start codon.

Prepare the 4X master mix. Use the protocol in the 4X column as a c...