In order to investigate the action of a newly discovered bacterial membrane protein that is a light driven proton pump, you purify the protein and assemble it with phospholipids into liposomes. In an attempt to determine the directionality of pumping, you create the liposomes so that they contain an indicator dye that is blue at high pH, colorless at neutral pH, and red at low pH. You then place the liposomes in an aqueous solution at a neutral pH and then expose them to light. To your dismay, the interior of the liposomes stays colorless. You believe that the bacterial protein has not been damaged by the purification and incorporation of liposomes. Puzzled, you go next door to find out how the other two groups of graduate students are doing on their problems.
Your colleagues in group two are working with the same protein. This group tells you that they have sequenced the protein and infer that it must be a five-pass transmembrane protein with amino and carboxy termini that extend well away from the membrane.
The third group has been studying the response of the same bacterial protein with the enzyme chymotripsin. It turns out that the only site that chymotripsin cuts in this protein is the second hydrophilic segment (counting from the amino terminus of the protein). When chymotripsin is added to intact bacteria it destroys the bacterial cellâs ability to pump protons.
In a flash of inspiration, you go back to your lab and add chymotripsin to your liposome preparation. When placed in the light, the interior of the liposomes now turns red.
2a) From the data given above, how did the second group infer that the protein was a five-pass transmembrane protein?
2b) What is the explanation for the reason why there was no color change in your original experiment? (Hint: Answer must explain why you got the observed results.)
In order to investigate the action of a newly discovered bacterial membrane protein that is a light driven proton pump, you purify the protein and assemble it with phospholipids into liposomes. In an attempt to determine the directionality of pumping, you create the liposomes so that they contain an indicator dye that is blue at high pH, colorless at neutral pH, and red at low pH. You then place the liposomes in an aqueous solution at a neutral pH and then expose them to light. To your dismay, the interior of the liposomes stays colorless. You believe that the bacterial protein has not been damaged by the purification and incorporation of liposomes. Puzzled, you go next door to find out how the other two groups of graduate students are doing on their problems.
Your colleagues in group two are working with the same protein. This group tells you that they have sequenced the protein and infer that it must be a five-pass transmembrane protein with amino and carboxy termini that extend well away from the membrane.
The third group has been studying the response of the same bacterial protein with the enzyme chymotripsin. It turns out that the only site that chymotripsin cuts in this protein is the second hydrophilic segment (counting from the amino terminus of the protein). When chymotripsin is added to intact bacteria it destroys the bacterial cellâs ability to pump protons.
In a flash of inspiration, you go back to your lab and add chymotripsin to your liposome preparation. When placed in the light, the interior of the liposomes now turns red.
2a) From the data given above, how did the second group infer that the protein was a five-pass transmembrane protein?
2b) What is the explanation for the reason why there was no color change in your original experiment? (Hint: Answer must explain why you got the observed results.)
