MCB110 FINAL Dec 13, 2007 . Your name and student ID QUESTION POINTS 1 (15 points) 2 (15 points) 3 (15 points) 4 (20 points) 5 (25 points) 6 (20 points) 7 (20 points) 8 (20 points) 9 (25 points) 10 (25 points) 11 (6 points) 12 (10 points) 13 (34 points) 14 (50 points) TOTAL (300 points) WARNING: Your exam will be taken apart and each question graded separately. Therefore, if you do not put your name and ID# on every page or if you write an answer for one question on the backside of a page for a different question, you are in danger of irreversibly LOSING POINTS!MCB 110 Page 2/15 Fall 2007 Final Name___________________________________ SID _______________________ Q1 - – Using the hydropathy plot below, do you believe the protein under study is an integral membrane protein? Why? (5 pts.) What can you predict from the plot in terms of secondary structure and why? (10 pts) The plot indicates that this is an integral membrane protein with a number of transmembrane helical segments, indicated by stretches of 20-25 amino acids with high hydropathy index. Three such segments are very obvious at around residue 100, 200 and 390, with additional ones at about 150, 230, 270 and 340 (this could be a seven helix transmembrane, G-protein coupled receptor – 5 extra points for this). Because 3 transmembrane helices are much more certain than the other 4, I would not penalize very severely those that just mention 3. The ideal answer will mention that 4 are likely but not clear.MCB 110 Page 3/15 Fall 2007 Final Name___________________________________ SID _______________________ Q2 – What methodology would you use to determine the if a GPI-anchored protein that has been expressed as a GFP fusion is able to diffuse on the membrane? (5 pts) Describe concisely how the experiment will be carried out, and the possible outcomes. (10 pts) FRAP - A defined region on the plasma membrane would be irradiated with a strong laser to photo- bleach the GFP. The intensity in this area as a function of time will then be measured. The plot of this intensity will start close to zero and then will increase until it reached a steady state plateau if the protein can diffuse (where how fast the recovery is obtains carries information on the diffusion coefficient), while it will remain close to zero if the protein cannot diffuse. Q3 – The Ca2+ concentration in the cytosol is about 105 times lower than in the endoplasmic reticulum. What is the initial free energy of movement of Ca+2 through an IP3-gated channel? In your calculation you can ignore the term concerning voltage potential. Can you explain why? (15 pts.) (2.3 RT = 1.4 kcal/mol at 25 ˚C) G = 2.3 RT [Cc]/[Cer] = 1.4 x (-5) = - 7 kcal/mol It makes sense to ignore the voltage component as the concentration gradient is so high and there is no significant voltage across the ER membrane.MCB 110 Page 4/15 Fall 2007 Final Name___________________________________ SID _______________________ Q4 - Can you describe three main differences between the molecular mechanisms of a glucose facilitative transporter (or uniporter) and the glucose-Na co-transporter (10 pts) What is the role of these two transporters in the movement of glucose across an intestinal brush border cell? (10 pts) 1- the uniporter moves glucose down its concentration gradient, the symporter moves it against 2 – the uniporter does not require the presence of a Na gradient 3 – the uniporter can move glucose in whichever direction is defined by the gradient, while the symporter works only in one direction. (I did not agree with Seemay on her objection, as I think that 1 and 2 are distinct and the students should point both out) The simporter brings glucose from the lumen of the intestine into the cell, creating a large concentration gradient of glucose. The uniporter lets glucose out of the cell and into the blood stream, in this case down a concentration gradient.MCB 110 Page 5/15 Fall 2007 Final Name___________________________________ SID _______________________ Q5 – Describe the events leading to the depolarization of a postsynaptic cell from the moment the action potential reaches the axon terminal of the presynaptic cell (15 pts). What is an important process that needs to be carried out at the synaptic cleft in order to reset the system and in which two ways can it be achieved (clue – cocaine interferes with this process)? (10 pts) Voltage-gated calcium channels are open letting ca into de cell. This leads to fusion of vesicles loaded with neurotransmitter to the presynaptic plasma membrane and the dispersion of the neurotransmitter into the synaptic cleft. The neurotransmitter binds to ligand-gated Na channels in the postsynaptic cell, opening the channel and leading to the entrance of Na into the cell and thus to its depolarization. The neurotransmitter needs to be cleared from the synapse. This is done either by breaking it down or by reuptake into the presynaptic cell via endocitosys.MCB 110 Page 6/15 Fall 2007 Final Name___________________________________ SID _______________________ Q6 – For the two integral membrane proteins depicted in the figure, indicate the succession of signal sequence, signal-anchor sequences and/or stop-transfer sequences that would give rise to their topology in the plasma membrane (20 pts). N-terminus …. SAII …. C-terminus N-terminus …. SAII …. STA …. C-terminus (Concerning Seemay comments, this is actually the only possibility that I can see. In both cases there is a big cytosolic N-terminus domain, and the ribosome will need a signal sequence to be bound by the SRP and taken to the ER (so in both cases there is an initial SAII – the +++ are OK if they include them in the right place –in front of the SAII, not behind!!!–, but if they do not mention them it is totally fine). For the second protein a STA then has to follow in order to define a second transmembrane region and then allow for the rest of the protein to be produced back in the cytosol). Notice the orientation!!! The big domains are the ones produced in the cytosol, the top ones are the one going into the ER, as they are the ones that finish up outside of the cell. The reason this question has that many points is because it puts together two different concepts, the topology when engaging the ER, and the fact that this is maintained all the way
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