Post UW 2B

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Apr 3, 2024

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Name: Jade Mair Douglas BIOL 200 2021W Post-Unit Worksheet B: Unit 2 (based on Problem Set 2.3.7) Let’s look at an experiment that uses SDS-PAGE to analyze membrane proteins. Recall that SDS-PAGE allows for proteins to be separated on the basis of size. This experiment focuses on proteins that have been modified by glycosylation (i.e. addition of sugar molecules). There are two types of glycosylation for membrane proteins: O-linked and N-linked glycosylation. O- and N- linked refers to the addition of sugars to oxygen or nitrogen atoms on amino acids, respectively. Experimental Set-up: Low-density lipoprotein receptor (LDLR) is a transmembrane protein responsible for the uptake of cholesterol into cells. Here, Pederson et al. (2014) examine the structure of LDLR through digestion with glycosidases (relatively large enzymes that remove sugar units). Glycosidase A removes O- linked sugars, and Glycosidase B removes N-linked sugars. Glycosidases were incubated with intact cells before the proteins were extracted and examined through SDS-PAGE. The results can be seen to the right. To help you get started:  What do the +/- symbols located above each lane indicate? What does that tell you about which glycosidases were added to each treatment group?  Recall what the relative position of bands on a gel indicates about the size of that protein. (In this question, the thickness of the bands is similar across all lanes, but what does the thickness of bands on SDS-PAGE gels generally indicate?)  Why do you think we specifically mention that we use intact cells for this experiment? Questions: a) Describe what the data show for each lane and explain how LDLR was affected by each treatment. Each lane represents a different treatment of the cell and therefore glycoprotein. column 1 represents the control (no glycosidases added), column 2 represents the addition of glycosidase A, column 3 shows results after the addition of glycosidase B, and column 4 of both glycosidases. The data show that the size of the proteins decreased linearly across treatment columns, i.e. treatment 1 had the largest proteins at ~98 kDa and treatment 4 had the smallest proteins at ~85kDa.

Name: Jade Mair Douglas BIOL 200 2021W b) Based on the information provided about LDLR in this question and on the above data, what can you conclude about the structure of LDLR at the plasma membrane? Based on the information provided, it can be concluded that the structures of N- linked sugars are larger than those of O-linked sugars. This is because in treatment with glycosidase B, which removes N-linked sugars, the size of the protein decreased more than after treatment with glycosidase A, which removes O-linked sugars. c) Draw what LDLR would look like in the cell membrane and label different components of this membrane protein. Mark where you would predict that the 2 enzymes (Glycosidases A and B) would bind LDLR to remove sugars. Make sure to label which side of your membrane is intracellular and which is extracellular.

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