Chem 4110 Fall 2012 Homework #5 Protein Data Bank and Analysis of Protein Structures

60 points; Due Wednesday, Oct. 10^th Make sure to address all questions in red;bold

Go to the PDB website: http://www.rcsb.org/pdb/home/home.do

I would encourage you to browse around the site and check out some of the cool tools/links and interesting information.

For the assignment, click on the link: http://www.rcsb.org/pdb/101/motm.do?momID=154

to explore the eight enzymes of the citric acid/Krebs/TCA cycle, that is central to cellular metabolism.

Specifically, we’ll be focusing on Succinate Dehydrogenase, considered the sixth step in the Krebs cycle.

Click on: http://www.rcsb.org/pdb/101/motm_disscussed_entry.do?id=1nek

Scroll down the page, and note the sequence display showing locations of secondary structural elements and connecting segments. This information is taken from an experimentally-determined structure and is specific and accurate compared to the level of accuracy of predictions based solely on the helical/sheet propensities of amino acids. Click on the figure to see higher resolution.

Go back and scroll down to see various ligands and how they interact with this protein. It is instructive to note that this enzyme is the only member of the citric acid cycle to be a membrane protein and is located in the inner mitochondrial membrane, projecting into the matrix space. This enables it to funnel the electrons into a pool of freely-diffusible ubiquinone, thereby reducing it to ubiquinol which can power complex III, continuing the electron transport chain. Notice the membrane lipids, the redox cofactors: iron-sulfur cluster, FAD and ubiquinone, and oxaloacetate which likely binds as a substrate-mimic, thus labeling the location of the active site when we begin to investigate the molecular details of this enzyme…

Type the PDB identifier: 1nek into the search window on the PDB home page.

Answer the following questions in a Word document, including your name, and all remaining segments of this assignment:

1. What technique was used to solve the protein structure?

2. What is the resolution limit of the data?

3. What is the R-factor (R-Value) for this structure? Briefly discuss whether this is a reasonable value or not. Also address one reason we might expect this to be a little higher than average for this structure.

4. How does the R-free compare? Explain why this reasonable or not.

5. Based on these three parameters, briefly state your level of trust in the accuracy of this structure.

Now, go to the download files link on the left navigation bar: http://www.rcsb.org/pdb/download/download.do

and download the file (1nek) in PDB format (deselect mmCIF format), and uncompressed works just fine for nearly all files. Save it to a folder location that you can easily find and access for use with PyMol.

Installation of PyMol:

Go to the G drive on the SUU computer network. Select the following folder:

G:\Classes\Howard\Chem4120\pymol-0_99rc6-bin-win32

Double click on SETUP.EXE

select place you want to install the program. On a school computer, you may need to make sure you install it in a place you have write-privileges enabled. Maybe an F-drive or other personal drive. If you have a PC operating windows, this version should work fine.

We should all have access to PCs on campus. If you prefer a mac, you’ll need to go to the PyMol website, and register as a student academic user to download a version that will work on OSX. Linux fans, there is a version for us as well.

Once you specify where to install it, click install, and then upon completion, select ‘yes’ to make it the default pdb-viewer. This way, double-clicking on a pdb file (or PyMol log file), will use PyMol directly.

To make sure it’s working, go to this folder:

G:\Classes\Howard\Chem1120\Proteins

and double click on the “rhodopsin_log.pml” file icon. PyMol should open and there should be an alpha helix visible in the middle of the screen in stick form. (this is the file I used in class to look at helices, and how they fit together to build the structure of rhodopsin). If PyMol is working properly, you should be able to select various objects, and render them in different styles.

Now, for the assignment, open the pdb file you downloaded for succinate dehydrogenase (1nek).

The molecule should appear on a black background as a line structure with carbon atoms green, nitrogen blue, oxygen red, sulfur yellow, etc. by default. Press the left mouse button and rotate the molecule by moving the mouse around.

How many different polypeptides are there in this protein?

A quick way to see, along with how they are arranged in the quaternary structure select the color tab next to the Succinate_Dehydrogenase _1NEK object (top right of the screen). Left click, and select color by spectrumà rainbow. Now each chain (which is denoted by a different letter as a chain-identifier in the pdb file) is colored differently. By rotating it around you can get an idea of how these polypeptide chains fold up and fit together.

Still, it is difficult to see the folds within each domain when it is all lines…

Create a separate object including all atoms in the pdb. This will allow us to toggle these cartoon ribbon backbones on and off in the future, as desired:

create cartoon, /Succinate_Dehydrogenase_1NEK////

(This of course assumes you’ve renamed the pdb file to include the name as I have. modify the object name to be whatever you have specified for the pdb filename in your folder.)

Now turn off the original object by clicking it. Nothing looks to have changed, since the two objects are currently identical. Select the ‘H’ button next to the object named ‘cartoon’, and go down to ‘everything’ and let up. Everything should have disappeared (H=hide). Now click on the S(show) button, and scroll down to ‘cartoon’ and let the button up. Now, the backbone traces are visible, and you can see which regions are helical, and other secondary structural elements and how they fit together.

It’s even better to make each individual chain into a separate object, so it can be studied without the interference of other chains that may obscure what we want to see or display. So, let’s click on an atom within each chain to generate the atom “address” in the control window. This way we will have an address of the chain to specify for each chain object

I first clicked on the green chain and get this: You clicked /cartoon//A/THR`181/CA

So, I’ll create an object called “chain_A” with this command:

create chain_A, /cartoon//A//

Now I have an object listed in the upper right hand corner of the screen labeled chain_A.

I can now turn off the cartoon object, and now only the green polypeptide cartoon should be shown. And the program should have automatically centered and modified the “slab” thickness appropriately. (and if you have a (sele) object you don’t want, click the A button and select the delete option).

Create a new object called “oxaloacetate”:

create oxaloacetate, /Succinate_Dehydrogenase_1NEK///OAA/

Now we can see where it binds to the protein, and look at interactions that hold it there.

Create an FAD object:

create FAD, /Succinate_Dehydrogenase_1NEK///FAD/

A 2x2 iron sulfur cluster:

create Iron2Sulfur2, /Succinate_Dehydrogenase_1NEK///FES/

And one for the 4x4 cluster:

create Fe4S4, /Succinate_Dehydrogenase_1NEK///SF4`303/

And one for the 3x4 cluster:

create Fe3S4, /Succinate_Dehydrogenase_1NEK///F3S`304/

And one for the heme group:

create heme, /Succinate_Dehydrogenase_1NEK///HEM`305/

And one for the cardiolipin lipid molecule:

create cardiolipin, /Succinate_Dehydrogenase_1NEK///CDN`308/

And one for the phosphatidylethanolamine lipid molecule:

create P-ethanolamine, /Succinate_Dehydrogenase_1NEK///EPH`309/

And last, but not least the ubiquinone molecule:

create ubiquinone, /Succinate_Dehydrogenase_1NEK///UQ2`306/

Now, just a few more (one for each other polypepetide chain):

create chain_B, /cartoon//B//

create chain_C, /cartoon//C//

create chain_D, /cartoon//D//

Now, we are all done making objects!! But feel free to make as many more as you wish, depending on what you want to look at in detail.

Use this now-user-friendly session of Pymol to investigate this incredibly-cool enzyme. Note how the structure fits together with protein secondary-structural elements. Also how the redox cofactors are bound, and arranged to allow the conduction of electrons from one to another along the chain. Electrons are extracted as a hydride ion from succinate to produce fumarate (alkane to alkene oxidation). The hydride with its pair of electrons, must be converted to two one-electron transfers to pass down the redox “wire”, ending up reducing ubiquinone to ubiquinol with two single-electron transfers (and picking up two protons from the matrix side of the membrane). For the assignment answer the following questions. It will be very useful to color the objects according to element type, choosing your preference for carbon (click Color button next to objectà color by elementà preference)

Also, to make measurements, click the ‘Wizard’ pulldown menu in the control window, and select ‘measurement’.

6. Give the distance in Angstroms between the closest atom of the oxaloacetate molecule, and the Nitrogen of the FAD molecule (which accepts the hydride upon oxidation of succinate, the actual substrate).

7. Measure distance from this nitrogen atom to the closest atom in the iron-2, sulfur-2 cluster. How does this compare with the first distance measured? Can an electron jump that far (Read discussion in first paragraph on page 593 in our textbook? (p. 607 In third edition)

8. List the edge-to-edge distances between the remaining elements of the redox chain, including the final acceptor ubiquinone

9. Is the heme molecule in the line of redox cofactors that conducts electrons from succinate to ubiquinone? What role, if any, do you think it serves in this process? Explain your reasoning.

10. Which of the four subunits of this protein do you think forms the transmembrane unit which anchors the complex in the membrane? Explain why you think this is the case.

11. Characterize each of the polypeptide chains in this protein as alpha-helical, beta-sheet, or mixed alpha/beta.

12. Finally, submit an image printed out in color, showing the chain of redox cofactors in a way that is clear to you, and you would use to present this concept of a “redox wire” through a protein to others. You may choose whether or not to show proteins, but if you do, make sure to render them in a way that you can still see this redox chain. Hint, change the background to white to minimize ink use (in control window: DisplayàBackgroundàWhite).

Enjoy this program, and experience the utility in using this platform to explore protein structure and function. The more you use this program to study the various proteins we’ll be encountering in biochemistry, the better you’ll get and the deeper your understanding will be in each case. A truly powerful tool, especially given the vast wealth of molecular structure data we have in the PDB. Use it as a tool on a regular basis, and you’ll have a massive advantage compared to most of your peers in graduate and professional programs who may not think realistically about life on a molecular level. Such an intuitive understanding develops gradually, but can become just as solid of an intuition as you have with commonly encountered everyday objects. A perspective that can provide valuable insight that may allow you to make the connection in a particular diagnosis or to enable the invention and improvement of a new technology of the future.