Answer Happy

A hydrophobicity plot allows for a prediction of the number oftrans-membrane domains in an integral membrane protein. To confirmyour prediction, you can design an experiment using fusionproteins. Each fusion protein contains a region of the originalmembrane protein fused to an enzyme. The enzyme moiety can belocated at the N-terminus or C-terminus of the fusion protein (seefigure below). In each experiment, one of the fusion proteins isexpressed in bacteria and is inserted in the cell membrane as thefull-length protein would be. Bacteria have a periplasm locatedbetween the cell membrane and the cell wall (see figure below). Theenzyme found in the fusion proteins is active in the periplasm butnot in the cytoplasm of bacteria.
Questions
a) We assume that the folding of this proteinis the same in the bacterial membrane and the membrane of aeukaryotic cell. In a cell, which end (NH2 or COOH) of the studiedprotein is extracellular? Explain your answer.
b) What is the number of trans-membrane domainsin the studied protein? Explain your answers.
Cell wall Cell membrane Cytoplasm Bacteria Periplasm NH₂ No Yes No Yes Fusion proteins (the blue circle represents the enzyme) NH₂ NH₂ Fusion protein | Enzymatic activity FL1 FL2 T1 T2 NH₂ A A A A B B B B C C COOH T2 D D COOH T1 -COOH FL1 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 FL2