Structure/Function of Biomolecules (Chem 240) Comparing Neuraminidase to the period at the end of a sentence.

Transcription

1 Comparing Neuraminidase to the period at the end of a sentence. Measure the distance between two atoms on either side of the enzyme. Select both atoms and from the toolbar, choose... tools...structure analysis...distances...create Assuming a period is 0.3mm wide, how many neuraminidase enzymes would stretch across a period if lined up side by side? In the command line, type: close all Measuring the volume of the active site Chimera knows how to calculate the volume of an enclosed blob, but not how to calculate the volume of a crevice or pocket that's open at one end. So to find the volume of the active site we'll need to use the CASTp server (CASTp stands for "Computed Atlas of Surface Topography of proteins). CASTp finds crevices, figures out where the "edges" are, and then reports back on the volume. Neuraminidase has already been analyzed by the CASTp algorithm; all we need to do is get the data. Open up a new instance of Chimera (you don't have to close the original one) File...Fetch by ID... type 2bat into the space next to CASTp The structure appears along with the CASTp window. Each line represents a different pocket that CASTp identified in neuraminidase. Click a few lines in the CASTp window and describe what is being shown in the main display: Find the pocket corresponding to the active site and report the MS volume: Report the ratio of the size of this enzyme to the size of its active site. In the command line of both chimera windows, type: close all 31

2 Tamiflu (oseltamivir), a mechanism-based inhibitor of neuraminidase Neuraminidase catalyzes the breakdown of sialyl galactose, a disaccharide of sialic acid and galactose: Influenza needs this enzyme to infect other cells. The disaccharide coats the outside of the cells that influenza targets. But think of what happens after a cell is infected and the virus has multiplied...to spread, it needs to get out of the cell. But it s attracted to the disaccharide all over the outside of the cell...so it can't leave. It would be like the Millenium Falcon trying to escape from inside a Star Destroyer; won't happen unless somebody shuts down the tractor beam. But if the virus has a means of shearing the sialic acid from the cell surface (shutting down the tractor beam), it can escape in large numbers and spread to other cells. Neuraminidase is the enzyme that makes escape possible. And here's tamiflu: Tamiflu was designed to mimic the middle structure of the neuraminidase reaction (the structure being attacked by the hydroxide ion). Strictly speaking, this middle structure isn't a transition state; it's an intermediate. The difference is, while an intermediate may be unstable and is, by definition, part way between reactants and products, it possesses local stability. In other words there are energy barriers on either side of an intermediate, but a transition state represents the top of an energy barrier. 32

3 Draw an energy diagram that represents the reaction catalyzed by neuraminidase. Label the axes appropriately, and label the positions of E + S, ES, E-I (the intermediate), EP, and E+P. On the same graph, but using a dotted line, sketch the pathway the reaction would take if presented with tamiflu. The key thing to remember is that tamiflu is a very stable version of the positively charged intermediate. Without that positively charged oxygen, it cannot react in the same way that sialyl galactose reacts. Load the structure of neuraminidase and sialic acid in Chimera (2bat) Examine the 2bat structure... Zoom in on the active site, which should show only a few sidechains and sialic acid. Chimera has detected a ligand and has only displayed side chains within a few angstroms of that ligand. It's a nice way to highlight potentially important residues. For what we want to do next, it will be easier if we add hydrogens. So in the command line type addh It will ask you to clarify a few things we don't care about. For each residue in the popup window, choose "single" and "0". Then click OK. 33

4 Illustrate 5 interactions within the active site that appear to stabilize the ES complex. Draw the sidechains below, identify each by sequence number, and use dashed lines to illustrate any noncovalent interactions. Also identify the type of noncovalent interaction. Sialic acid is drawn below in the same conformation as it has in 2bat. It might also help to have Chimera detect possible H-bonds. Select sialic acid, then tools...structure analysis...findhbond check "Only find H-bonds with at least one atom selected" and click OK. Now in the same chimera window, in the command line, type open 2hu4 This structure contains several copies of the neuraminidase-tamiflu complex. Lets delete all but one. Using the toolbar... select...chain...a (2hu4) select...invert (selected models) actions...atoms/bonds...delete Now we'll fit the two over each other to compare their structures. Tools...Structure comparison...matchmaker Choose 2bat as the reference structure and 2hu4 as the structure to match. Click OK. Report the ID of the carbon atom in tamiflu that has replaced the oxygen of sialic acid. In this view you can see that the two ligands bind in very similar orientations. From this view, does it appear that neuraminidase makes more or fewer H-bonds to tamiflu than to sialic acid? 34

5 Tamiflu binds tightly in part because the enzyme "fits" around it a little better than it does to sialic acid. One residue that has clearly shifted is Asp 151. Measure the distance between the two Asp151 residues, using the furthest-separated oxygen atoms as reference points. Select both oxygens and from the toolbar, choose... tools...structure analysis...distances...create distance is Structure 3cl0 features a mutant neuramindase complexed with tamiflu. This mutant neuraminidase came from tamiflu-resistant strain of avian influenza (or H5N1) isolated from an infected patient. The mutant neuraminidase was crystallized with tamiflu bound. In the same instance of Chimera with 2bat and 2hu4 displayed, open 3cl0 (note...that's a small L and a zero on the end) Match 3cl0 with 2bat and 2hu4. (see previous page) The mutation is to residue 274, which isn't currently displayed. Let's show it. In command line: sel :274 then, on the toolbar: actions...atoms/bonds...show What is the mutation? Make sure residue 276 is showing by the same method as above. Provide an explanation for this strain of avian influenza's resistance to tamiflu. Keep in mind that Tamiflu can clearly still bind to the mutant neuraminidase, because otherwise this structure wouldn't exist. You might also try using CASTp to compare the active site volumes of 2bat and 3cl0. Written with inspiration from the Neuraminidase entry on the Molecule of the Month (David Goodsell) feature at RCSB.org 35

6 Exploring Carbohydrates One benefit of teaching labs in biochemistry is that you are presented with the opportunity to think about the structure of biomolecules in a different context that requires different modes of thinking and may therefore make the molecules more comprehensible. Historically, there have existed numerous assays designed to test for various properties of sugars. In this lab, we are going to explore some of these assays to study carbohydrates that we are likely to encounter in our diets. We will provide you with the reagents for various carbohydrate assays. They will be simple and quick to perform, so the majority of your laboratory experience will involve planning and analyzing your data, rather than performing your experiments. This is often true in biochemistry experimental work. You will be testing your own samples containing different types of carbohydrates. You will be provided with a series of questions at the end of lab. Take careful notes during lab, since you will be expected to include information about how and why you chose the samples to test, what hypotheses you were testing and what the rationale was for the test you preformed. Often researchers uncover something confusing during the course of their experiments and have to modify the test they need to perform to test their hypothesis. You will also want to include what each assay revealed about your samples and how your samples compare to any controls you ran. Make sure that you record all data since you will be reporting on how you analyzed your samples, including sample preparation. Finally you will have a chance to determine if your results support your hypothesis and what you concluded about your data. Prior to the lab period, you should plan your experiments and make predictions about your results. This will allow you to think on your feet during the lab period so that you can perform any additional experiments that are warranted to allow for a complete and intelligent discussion of your data. This will help you to avoid the need to include statements such as Next time, we could do an additional experiment such as. An important component of your experimental design should be controls: a standard for comparison. Based on your knowledge of the structure of carbohydrates, you should design positive and negative controls for each test you perform. The Assays: Reagents will be prepared for you. A general format for each assay is shown. You may decide to modify it. Barfoed s Assay Mix 1 ml of reagent with 5 drops of sample. Heat. Positive test for monosaccharides is the formation of a brick-red precipitate within five minutes. (Disaccharides generally do not react even within ten minutes). Reagent: 70 g copper acetate monohydrate 9 ml glacial acetic acid to 1 liter with water 36

7 Benedict s Assay Mix 1 ml of reagent with 5 drops of sample. Heat. Positive test for reducing sugars is the formation of a precipitate within five minutes. Color ranges from green to yellow to brick red (red indicates the most reducing sugar). Reagent: 100 g sodium carbonate 173 g sodium citrate dihydrate to 850 ml with water; mix add 17.3 g copper sulfate pentahydrate in 100 ml water bring the total to 1 liter with water. Seliwanoff s Assay Mix 1 ml of reagent with 5 drops of sample. Heat. Positive test for ketohexoses is a change to an orange/red color. (Aldohexoses will turn light pink, but it takes longer). Reagent: 1 g resorcinol in 330 ml concentrated HCl to 1 liter with water Iodine Assay (polysaccharides) Add 1 drop of iodine solution to 5 drops of sample. A positive test for starch is a dark blue-black color. Other polysaccharides react to a lesser extent to form a red-brown or reddish-purple color. The Samples: Provided for you: Table sugar, Splenda, Sweet-N-Low, lactaid, maltose, glucose, fructose Provided by you: Any products that contain sugars or sugar-substitutes. Note: Any food items brought into a lab environment are considered contaminated and are no longer suitable for consumption. Procedure: Perform all of the available assays with the provided samples as well as with any samples you brought to the lab. Consider the following prior to lab: Can you compare the relative amounts of sugars in the different samples? How will you prepare the samples? How will you heat the samples? (A boiling water bath? A heat block?). What samples will you test? (Controls? Different concentrations? Unknowns?) What data will you record? (Precipitate? Color? Time?) 37