bioinformatics of green fluorescent protein · 2010-03-09 · page 5 protein visualization using...

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Bioinformatics of Green Fluorescent Protein

This bioinformatics tutorial explores the relationship between gene, protein, and biological function in the context of the Green Fluorescent Protein (GFP). In this tutorial you will learn how to find a protein using simple and advanced search tools on the RCSB PDB website, and will use molecular visualization tools to explore the GFP structure and find the relationship between structure and function. You will explore the properties of primary, secondary, tertiary and quaternary structure. Finally, you will find the GFP gene and view important mutations and explore the changes that occur in both protein structure and biological function.

Please send any comments or questions about this tutorial to [email protected].

I. Bioinformatics: Computation, Databases and Visualization Find the Green Fluorescent Protein by keyword search on the PDB website.

1. Go to the RCSB PDB website at http://www.pdb.orgi 2. Perform a “keyword” search by typing a keyword in the text box on the search bar at the top of

the first page: Search for “green fluorescent protein”

3. Click the Search button. 4. The result page will contain a list of proteins that have something to do with GFP. For this exercise, we will use a protein that was taken from the jellyfish Aequorea victoria with PDB ID 1EMA. You can easily find this protein by sorting the search results by PDB ID.

1. Click on the Sort link on the left

side of the browser NOTE: If you do not see the link to Refine Search then click on the Results tab at the top of the control panel. Make sure this tab is the active tab. 2. Click on PDB ID to sort by PDB ID

Other ways of limiting your search results to find a structure include selecting the Refine this Search option from the left menu or sorting by release date to find the most recent structure. Explore the GFP structure 1. On the results page click on the image that has the title 1EMA.

2. The web page that is returned is called the Structure Summary page. On the right side of this page you will see a box containing an image and a series of links to Display Options. These are the 3-D molecular viewers.

3. Click on the link called QuickPDB. This is a Java molecular viewer that will download and display the 3-D coordinates of this structure.

Visualize the protein sequence. After QuickPDB is launched, you should be able to see the following:

1EMA’s amino acid sequence is displayed in the top box and the structure of the protein is in the bottom box. The molecular viewer structure only shows the three-dimensional path that the amino acids make when they are linked together. This path is called the polypeptide chain. Each amino acid in the chain is linked to the next by a strong bond, called a peptide bond.

1. Click and drag the structure in the bottom structure viewer (bottom box) to rotate the structure. 2. Roll your mouse over the sequence viewer (top box) and watch the Residue label change. The

sequence of amino acids shown in this box is that we call the primary structure of the protein.

The amino acid sequence is typically represented in bioinformatics as a one letter ID – Glycine is ‘G’ and Alanine is ‘A’. For a complete list see Appendix I.

3. If you move the mouse over the amino acid sequence listed in the top box, you will see the Residue Label change in the top box, while that residue is highlighted in the structure view in the bottom box. In the example pictured above, the residue F64 is highlighted.

4. Click and drag over the residues to select them. For this example, click and drag over F 64 and

V68. ** If you make a mistake, click on the Reset button at the bottom of the screen. This will reset the structure to show all amino acids in green. 5. After these amino acids are selected, they will appear blue in the 3-D structure window below. It

should look like the following:

The blue amino acids show the location of what is called the chromophore. This is the light harvesting area of the protein. Now let’s take a look at the GFP structure in more detail.

Protein Visualization using Protein Workshop 1. Click the back button on the web browser. 2. You should now be back at the Structure Summary page.

a. If you can’t get back to the structure summary page simply enter the text, “1EMA” into the top query bar and click the Search button.

3. Under the image click on the link that says Protein Workshop 4. This will download the viewer. In the process of doing this it will ask you if you want to trust

this application. This is part of the Java security mechanisms; you simply accept/trust each one and click run when prompted.

Once the structure is loaded you should see something that looks like the following:

The first thing to notice is the overall layout. The viewer is to the left and the control panels are to the right. If you click and drag the mouse in the viewer you will see the structure rotate. For full descriptions of all the controls in the viewer please refer to the appendix II. The second most important thing to note is the layout of the control panel itself. This application was designed for quick and simple editing in a step-by-step process. Notice the numbered boxes on the panel: 1, 2, 3, 4 (these are highlighted in the above figure). This is to help you go through the steps of using this tool.

Boxes 1-3 change when the Tools, Shortcuts, or Options buttons are selected:

Tools Menu Shortcuts Menu Options Menu

What can we see with Protein Workshop?

What we are currently looking at is what is called the “ribbon” representation of the protein structure. Much like the previous viewer, this representation forms a line (like a ribbon) that represents the polypeptide chain of the primary structure. Once again, we are simply looking at the chain of amino acids that has folded into a 3-D object. Why do we look at structures this way? Why do we use the ribbons? Let’s turn off ribbon view and view all the atoms of the protein: Following the 1-2-3-4 step process in the Control Panel under the Tools menu: 1. Click on the Visibility Button 2. Select Ribbons 3. Select Toggle the item to appear/disappear 4. Click on 1EMA (the PDB ID) in the bottom tree viewer.

At this point the viewer should be blank. Let’s do the same thing, but we’re now going show all atoms. 1. Click on the Visibility Button 2. Select Atoms and Bonds this time. 3. Select Toggle the item to appear/disappear 4. Click on 1EMA (the PDB ID) in the bottom tree viewer.

You should see the following:

Each dot is an atom. The lines between these atoms are bonds. The colors of the atoms are as follows: Green is Carbon Blue is Nitrogen Red is Oxygen You can find the entire color scheme in the appendix.

Zoom into the protein Let’s get a better look at the protein structure with all atoms and bonds shown. To do this we are going to zoom into the protein. Hold the shift key down. Click and drag the mouse downward (For more information on viewer interaction see the appendix II at the end of this document. This will bring the protein closer. Can you find the chromophore? (Hint: look for yellow/orange) As you can see, this representation shows a lot of information and it’s difficult to find specific structural features when all atoms are shown. To return to the default view of the structure, we’ll follow our steps in reverse.

1. Click on 1EMA in the bottom tree viewer. This will make the item disappear. 2. Select on “Ribbons” in box #2. 3. Click on 1EMA in the bottom tree viewer. This will make the item appear, but now in the

ribbon representation. Combine ribbon view with atom view If we want to see how specific atoms interact with other parts of the protein, we need to be able to see secondary and tertiary structure as well as atom level detail. This requires combining both atoms and ribbons. Activity: Draw the chromophore using atoms and bonds while keeping the ribbon structure visible.

1. Select the Visibility tool 2. Select Atoms and Bonds for what you want the tool to affect – This means that when we

select the chromophore, it will appear as atoms bonds.

3. Select Toggle the item to appear/disappear. This will help to see where the chromophore fits in the entire structure.

4. In the tree box, open up Chain A. Scroll down and select the position of the chromophore – CRO 66. Selecting and deselecting will cause the chromophore to appear (in atom representation) and disappear. You can rotate the structure to see how this piece fits in the overall shape of the protein.

Here, the chromopore is not shown Here, it is shown in atoms and bonds

II. Gene and Protein Sequence

Look at primary structure using the UniProt The UniProtii database is a resource that organizes and annotates protein sequences. This database contains important information for researchers to study the relationship between protein sequence and protein function. There are two sides to this database. One annotates sequences using a staff of scientists while the other is an automated process using sophisticated software tools. We will use the human-annotated version called Swiss-Protiii. Why do you think the database is designed this way? Why isn’t the entire process automated? Uniprot takes in about 800,000 sequences per year and this number is growing exponentially. 1. Point your web browser to the following URL: http://ca.expasy.org/ 2. In the top search box select Swiss-Prot/TrEMBL and enter the text “Green fluorescent protein”

3. Click on the Go button. You should see a result much like the following:

You can ignore the bottom section of the results labeled UniProtKB/TrEMBL; this is the computer-generated side of the database. Of the results in the UniProtKB/Swiss-Prot click on the one titled GFP_AEQVI(P42212) You should now see the summary page for GFP.

Shortcut: To jump to this page enter the accession id “P42212” into the search window on the site http://ca.expasy.org. Click the Go button.

Let’s take a look at the gene sequence.

1. Scroll down to the section titled Cross-references

2. Click on the GenBankiv link on the top line. 3. On the resulting page, scroll down to the bottom. This is the gene sequence that translates the

protein sequence of GFP. Do you notice something strange about this sequence? Does this sequence translate directly into our GFP? It turns out that there is more information here than we need? Only parts of this sequence are used for the protein. Let’s simplify this by looking at only these coding regions.

Shortcut: To jump to this page, go to the NCBI website: http://www.ncbi.nlm.nih.gov/ and select the Nucleotide database in the search pulldown menu. Enter the text M62654 and click the Go button.

4. Click on the link labeled gene under the FEATURES category.

5. Scroll to the bottom of the page. This is the entire DNA sequence that translates into our protein GFP.

Translation of the DNA sequence to the Protein Sequence Now that we have this nice DNA sequence let’s translate it back to the protein sequence using the translate tool provided on the expasy web site. The first thing we need to do is copy the DNA sequence to your computer clipboard. We will then paste this into the tool.

1. Scroll to the top of the sequence page and locate the menu option called Display. From the pulldown menu for Display, change GenBank (Full) to FASTA

In the resulting page select everything except the first line. In other words we just want to select the actual sequence. (The FASTA format always has a comment line on the top line and all subsequent lines are the sequence).

2. Copy this to your computer clipboard using Edit->Copy from the Browser menu or for Mac users: ‘Apple’ + ‘c’, and for PC users: ‘ctrl’ + ‘c’

3. Point your web browser to the Translate tool: http://www.expasy.org/tools/dna.html

4. Click in the text window and paste your sequence using Edit->Paste from the Browser menu or

for Mac users: ‘Apple’ + ‘v’, and for PC users: ‘ctrl’ + ‘v’ Your browser should look like the following:

Warning: Notice that the first line starts with “GATAAC…”. If your first line starts with “>gi|1555662… “, you forgot to take out the comment line of the FASTA format. You only want to copy and paste the sequence.

6. At the bottom of the page, change the Output format choice to “Includes Nucleotide Sequence”

7. Now click on the Translate Sequence button.

The results will show six different sequences that each represent the different reading frames of DNA (three in one direction and three in the other). Let’s take a moment and look at these results.

This shows how the protein is translated. Each line contains the DNA sequence and highlights the three-letter codon along with the corresponding amino acid. If we look at the sequences labeled 5’3’ Frame 2 we will see that ‘g’ is not used, ‘ata’ translates to ‘I’, ‘aca translates to ‘T’, aag translates to ‘K’ and ‘atg’ translates to ‘M’. We can verify this by look at the genetic code table and replacing the U’s with T’s (see appendix III). The start codon is AUG (or in the DNA case it is ATG). This means that the process of translation, where the mRNA sequence is converted into a protein sequence, requires this three-letter code in order to start. This start codon occurs pretty early in our sequence (11th from the beginning), so this is probably a good result. 8. Click the link titled 5’3’ Frame 2. The result page highlights the Methionine residues, or the starting point of the protein sequence. This corresponds to the ATG discussed previously. Click the first ‘M’ in the sequence. The resulting page will have a sequence that looks a lot like the protein sequence we started with. How can we tell if this sequence is the same as the one we had before? Compare a protein sequence with other protein sequences in a large database What can we tell from knowing the sequence of amino acids in a protein? If two proteins have very similar sequences what does that say about the structure and function of that protein?

From the result page in the translation tool click the link that says Fasta format (highlighted in blue letters). You should get the following result: >virt|VIRT14468|VIRT_14468 Translation of nucleotide sequence generated on MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL VTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFYKDDGNYKSRAEVKFEGDTLV NRIELKGIDFKEDGNILGHKMEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAD HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMILLEFVTAAGITHGMDELYK Now let’s use this protein sequence and compare it with sequences in the Protein Data Bank. If we find sequences that are the same that means that a researcher somewhere in the world solved the 3-D structure of this protein. 1. Copy the sequence (remembering not to copy the first line) 2. Go to the RCSB PDB website: http://www.pdb.org 3. On the right of the top blue query bar click the Advanced Search link

(You can also get to the Advanced Search by clicking on the Search menu on the list of menu options on the left side of your web browser.)

4. On the advanced search window click on the Choose a Query Type and select the option labeled, Sequence (Blast/Fasta)

This will cause the user interface to change to allow you to enter parameters for perform this search.

5. Click inside the box titled Sequence and paste your sequence from the translation tool. It


6. Now click on the Evaluate Query button on the bottom right corner.

The results page will have a list of proteins in the Protein Data Bank that closely match the sequence you entered. You can see the similarity by looking at the sequence alignment viewer for each structure:

As you can see, there are several structures in the PDB that contains similar sequences. In fact, the structure labeled 1GFL is almost identical, with just five letters in the entire sequence that are different (highlighted in orange). What does this tell you about the structure of the protein you used for the search? Do you think this will have the same structure as 1GFL? What about the chromophore? Is this the same? If we know that the structure 1GFL has a functional chromophore can we assume that this is the same with the query sequence we found in the Swiss-Prot database?

III Protein Folding Secondary structure We have explored the primary structure of GFP and we know that it is simply the sequence of amino acids linked together by a strong bond (covalent bond) called the peptide bond. We know that the order of the amino acids in the sequence contributes to the shape of the three dimensional protein. Secondary structure is the first level of molecular folding and consists of mostly alpha helices and beta sheets. Hemoglobin is a molecule made up of several alpha helices with no beta sheets whereas silk and a protein called fibronectin, are made entirely of beta sheets. Let’s look at hemoglobin using the RCSB PDB website (www.pdb.org): 1. In the top search bar enter the PDB Id “4HHB”

2. Now click on the tab on the menu bar that says Sequence Details

3. You are should see something that looks like this:

This view shows the secondary structure of hemoglobin next to the primary structure (the protein sequence). Notice the squiggly lines. These are the alpha helices that we saw in the structure. Hemoglobin has only alpha helices and “turns”.

Now take a look at fibronectin. This protein is found in the blood plasma and plays an important role in wound healing.

1. Enter the PDB id for fibronectin: 1FNA

2. Click on the Sequence Details tab at the top of the Structure Summary page.

3. You should see the following:

Notice the arrows. These are beta strands. As you can see there are no alpha helices.

Let’s take a look at a protein Green Fluorescent Protein.

1. Enter the PDB ID: 1YFP in the top search bar on the pdb web site:

2. Click on the Sequence Details tab

3. You should see the following sequence with secondary structure:

Notice this structure has both alpha helices and beta strands. The chromophore is located in at position 66 and is labeled with an “x”.

Visualize details of secondary structure using Protein Workshop

Let’s take a look at the secondary structure in more detail. A strong bond, called the peptide bond, links the amino acids in the protein sequence together. The secondary structure is held together by series of weaker bonds called hydrogen bonds.

A hydrogen bond occurs between certain atoms on the main chain of the protein. We won’t get into the details of the chemistry but an important property is that the bond distance is about 2.5 to 3 Ångstroms. We can measure this if we know which atoms are involved in the bond. To do this let’s look at the chemical structure of an amino acid:

The carboxylic acid group (red) of one amino acid hydrogen bonds to the amine group (blue) of another amino acid if the distance is within around 3 angstroms.

We can see this if we look closely at the atoms involved in secondary structure formation. Go to the RCSB PDB website (www.pdb.org), and search for the GFP structure with PDB ID 1EMA. Launch the Protein Workshop program using the link located under the image of the structure on its Structure Summary page. Once this loads, move the structure so it appears large in the window. To measure the hydrogen bonding from one strand of the beta sheet to the other: 1. In the viewer window click on two strands that are next to each other. (Do not do anything in

the control panel). You should see the following:

The structure above has the ribbons and atoms displayed together. You may need to zoom for the next step.

2. Click on the Lines button in the control panel. Leave the other options. Your control panel


3. Now click on a blue atom (the amine) one strand and a red atom (carboxylic acid) of the other strand.

This will draw a white line with a number that represents the distance in angstroms.

You can see that the distances are around 2.7 to 3 angstroms. This is what stabilizes the secondary structure. Activity: Measure the hydrogen bond distance for bonds within an alpha helix. You can use hemoglobin ( PDB ID is 4HHB)

Tertiary structure

The tertiary structure is also called the protein fold. As mentioned before the “protein folding problem” is one of the most important problems in science today. Today there are new proteins with unique sequences that have no means of structure analysis. Scientists from around the world are finding new ways to organize and classify the structural information we do know in the hopes of discovering the principles of nature that dictate these folds. Many of these secrets have already begun to unravel. One interesting thing to note about protein folds is that similar folds can appear in proteins with unrelated function. Because of this there are databases that organize proteins in the PDB in a hierarchy of folds. The two main databases are called SCOPv and CATHvi. Let’s see how GFP is represented in these schemas. In the top search text box, enter the PDB id for GFP: 1EMA Scroll down to the bottom of the Structure Summary page until you see red words that say SCOP Classification and CATH Classification

There are many levels of fold classification and from this we can see that SCOP classification contains a Fold called GFP-like after Alpha and beta proteins (a + b). Judging by the name of this, it seems that GFP has a pretty unique fold. We can verify this by clicking on the GFP-like link. This automatically searches the PDB for other proteins classified by SCOP as GFP-like. When we do this we can see that these proteins all have the same function, that of green fluorescent protein. If we move up a level and search the PDB for all Alpha and beta proteins we can see that we have a functionally diverse result. One of the interesting things about the protein structures is how simple yet functionally diverse these proteins are. Activity: Browse the SCOP hierarchy on the PDB website using the browse tool.

1. Click on the search tab in the upper left corner of the web site. 2. Click Browse database 3. Click SCOP Classification

Quaternary structure The quaternary structure of a protein is when more than one protein (polypeptide chain) combine to form a larger protein unit. The most common example is Hemoglobin. Let’s use Protein Workshop to highlight the subunits of the quarternary structure of hemoglobin. 1. Go to the Protein Data Bank website: http://www.pdb.org 2. Enter the PDB ID: “4HHB” into the top query bar. 3. Open Protein Workshop 4. In Protein Workshop change the colors for all four chains listed in the sequence tree (panel 4).

The combination of these chains together yields the quaternary structure.

Appendix I Letter codes for amino acids in a protein chain: G Glycine Gly P Proline Pro A Alanine Ala V Valine Val L Leucine Leu I Isoleucine Ile M Methionine Met C Cysteine Cys F Phenylalanine Phe Y Tyrosine Tyr W Tryptophan Trp H Histidine His K Lysine Lys R Arginine Arg Q Glutamine Gln N Asparagine Asn E Glutamic Acid Glu D Aspartic Acid Asp S Serine Ser T Threonine Th

Appendix II Protein Workshop Viewer Interaction Rotate Click and drag left button Zoom Click and drag middle button. OR Shift + Right Click and drag Translation Click and drag right button OR Ctrl + Right Click and drag. There are two ways to select features in the structure. 1. Select a residue in the viewer by single-clicking on the structure. 2. Select using the selection tree. The selection tree includes selection of the structure, chain, residue and even atom level. For more information on Protein Workshop please visit RCSB PDB website at www.pdb.org Appendix III The genetic code

References i H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne: The Protein Data Bank. Nucleic Acids Research, 28 pp. 235-242 (2000). ii Bairoch A, Apweiler R, Wu CH, Barker WC, Boeckmann B, Ferro S, Gasteiger E, Huang H, Lopez R, Magrane M, Martin MJ, Natale DA, O'Donovan C, Redaschi N, Yeh LS (2005) The Universal Protein Resource (UniProt) Nucleic Acids Res. 33: D154-159. iii Gasteiger E., Gattiker A., Hoogland C., Ivanyi I., Appel R.D., Bairoch A. ExPASy: the proteomics server for in-depth protein knowledge and analysis Nucleic Acids Res. 31:3784-3788(2003) iv http://www.ncbi.nlm.nih.gov/About/ v Murzin A. G., Brenner S. E., Hubbard T., Chothia C. (1995). SCOP: a structural classification of proteins database for the investigation of sequences and structures. J. Mol. Biol. 247, 536-540. vi Protein families and their evolution - A structural perspective. Orengo CA, Thornton JM. (2005) Annual Review of Biochemistry. Vol 74. p. 867-900. Review. Acknowledgements This tutorial was written by Jeff Milton and edited by Monica Sekharan and Christine Zardecki for the RCSCB Protein Data Bank.

bioinformatics of green fluorescent protein · 2010-03-09 · page 5 protein visualization using...

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