1) Knowing that the thickness of a lipid bilayer is 3 nanometers, what is the minimum number of residues in a trans-memb

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1 Knowing That The Thickness Of A Lipid Bilayer Is 3 Nanometers What Is The Minimum Number Of Residues In A Trans Memb 1 (55.32 KiB) Viewed 50 times

1) Knowing that the thickness of a lipid bilayer is 3 nanometers, what is the minimum number of residues in a trans-membrane a-helix to completely cross a lipid bilayer? Show your calculation. 3 pts 2) The hydropathy index of a protein defines the hydrophobic character of a protein. To create a hydrophobicity plot, the sequence of a protein is divided in windows of 7 adjacent residues. For a window of 7 residues, a hydrophobicity plot shows the index of the residue in the middle of the window (residue #4). a) The hydropathy index reflet the free energy (AG) of transfer of an amino acid side chain from a hydrophobic environment to water. Does a hydrophobic region of protein have a negative hydropathy index? Explain your answer. 3 pts b) The hydrophobicity plot shown was determined for protein X. Assuming that this protein is an integral membrane protein, what is the likely number of transmembrane domains in this protein? Explain your answer. 3 pts Hydropathy index 0 Hydropathy index 10 3 10 c) Predict the number of transmembrane domains in the integral membrane protein Y based on its hydrophobicity plot. Explain your answer. 3 pts 100 150 200 250 50 100 50 50 Residue number 100 130 130 100 150 200 Residue number 250 3) A hydrophobicity plot allows for a prediction of the number of trans-membrane domains in an integral membrane protein. To confirm your prediction, you can design an experiment using fusion proteins. Each fusion protein contains a region of the original membrane protein fused to an enzyme. The enzyme moiety can be located at the N-terminus or C-terminus of the fusion protein (see figure below). In each experiment, one of the fusion proteins is expressed in bacteria and is inserted in the cell membrane as the full-length protein would be. Bacteria have a periplasm located between the cell membrane and the cell wall (see figure
below). The enzyme found in the fusion proteins is active in the periplasm but not in the cytoplasm of bacteria. Cell wall cell membrane Periplasm ▬▬▬ Cytoplasm Bacteria Fusion protein FL1 FL2 T1 T2 Fusion proteins (the blue circle represents the enzyme) B C NH₂ NH₂ NH₂ Enzymatic activity No Yes No Yes B B C COOH T2 D The activity of the enzymes was determined in bacteria expressing the two full length fusion proteins (FL1 & FL2) and the two fusion truncated proteins (T1 and T2). Note that each measurement of enzymatic activity was done in bacteria expressing only one of the four fusion proteins. The results of the experiment are shown in the table below. COOH T1 COOH FLI COOH FL2 a) We assume that the folding of this protein is the same in the bacterial membrane and the membrane of a eukaryotic cell. In a cell, which end (NH₂ or COOH) of the studied protein is extracellular? Explain your answer. 3 pts b) What is the number of trans-membrane domains in the studied protein? Explain your answers. 3 pts 4) A membrane has three main types of membrane proteins shown on the diagram below: Integral membrane proteins (A) are embedded in the lipid bilayer Peripheral membrane proteins (B) are non-covalently associated with integral membrane proteins. Amphitropic membrane proteins (C) associate with the membrane via direct lipid-protein covalent interactions. Your goal is to experimentally determine the type of membrane association of three proteins (P1, P2, and P3).
In each experiment, a membrane sample is subjected to the following steps: Experiment 1: The membrane fraction is treated with SDS, and the solubilized sample is analyzed by SDS-PAGE. SDS is a anionic detergents that solubilizes lipid membranes and proteins. Proteins are visualized by staining the gel with Coomassie blue. Experiment 2: The membrane fraction is treated with a sodium carbonate solution and then subjected to centrifugation to separate the supernatant (soluble fraction S) from the pellet (membrane fraction M). The high pH (pH 11) of the sodium carbonate solution promotes the dissociation of ionic interactions between protein and membranes. Fractions S and M are analyzed by SDS-PAGE. Proteins are visualized by staining the gel with Coomassie blue. Experiment 3: The membrane fraction is treated with phospholipase C and then subjected to centrifugation to separate the supernatant (soluble fraction S) from the pellet (membrane fraction M). Phospholipase C is an enzyme that cleaves the linkage between proteins and their lipid anchors in membrane. Fractions S and M are analyzed by SDS-PAGE. Proteins are visualized by staining the gel with Coomassie blue. The data are shown below. Experiment Experiment 2 10:0 Experimen: 3 a) Based on data from experiment 1, what can you say about the type of membrane association for proteins P1, P2, and P3? Explain your answer. 2 pts b) Based on data from experiments 1 and 2, what conclusion can you draw about the type of membrane association for proteins P1, P2, and P3 (integral, peripheral, amphitropic). Explain your answer. 2pts c) Based on data from the three experiments, what conclusion can you draw about the type of membrane association for proteins P1, P2, and P3 (integral, peripheral, amphitropic). Explain your answer. 2 pts 5) Some proteins synthesized in the cytoplasm of cells are transported inside mitochondria. You are trying to determine experimentally whether proteins PA and Pa are cytoplasmic or mitochondrial. You use an in vitro translation system to produced radiolabeled proteins (PA or Pa*). You incubate one of the radioactive proteins with a fraction of purified mitochondria (step 1). After an hour you add to the reaction the enzyme trypsin (step 2). Trypsin is a protease that can digest proteins into very small fragments. The trypsin treatment is stopped after 30 minutes, the digested reaction samples are treated with SDS-PAGE
buffer to denature proteins (step 3). After denaturation, the reaction samples are subjected to SDS-PAGE and the protein gel is placed in contact to an X-ray film to detect radioactive proteins. A radioactive protein in the gel will expose the X-ray film and appear as a dark band once the film is developed. In control experiments, mitochondria are treated with a drug that block their membrane potential. This treatment occurs before mitochondria are incubated with the radiolabeled proteins. The data are shown below. 1 2 3 4 5 6 I Content of lanes 1 to 6 • Lane 1: Positive control, sample of the radiolabeled protein PA*. This sample was not incubated with mitochondria or treated with trypsin. • Lane 2: PA* incubated with untreated mitochondria then with trypsin. Lane 3: PA* incubated with drug treated mitochondria then with trypsin Lane 4: Positive control, sample of the radiolabeled protein Pa*. This sample was not incubated with mitochondria or treated with trypsin. Lane 5: P₂* incubated with untreated mitochondria then with trypsin. Lane 6: Pg* incubated with drug treated mitochondria then with trypsin Based on these data, identify the mitochondrial protein (PA and/or Ps). Explain your answer. 6 pts