protein visualization

Overview of this Lab

Visualization of the molecular structures of proteins for hemoglobin and other diseases.

Brief Introduction to Hemoglobin

Hemoglobin (also spelled “haemoglobin”) is a protein found in the red blood cells (erythrocytes) of vertebrates, and it is responsible for transporting oxygen throughout the body. When it forms an unstable, reversible bond with oxygen, it becomes bright red; this oxygenated state is referred to as “oxyhemoglobin.” In its reduced state, hemoglobin is more of a purple/blue color. Asphyxiation occurs when there is an excess amount of substances, such as carbon monoxide, in the blood; this is because hemoglobin forms a semi-permanent or permanent bond with compounds instead of reversible bonds with oxygen.

When red blood cells die, hemoglobin is broken up. Iron is salvaged, transported to the bone marrow by proteins called “transferrins,” and then used again in the production of new red blood cells. The remainder of the hemoglobin forms the basis of a chemical called “bilirubin,” which is excreted into the bile, eventually reaching the intestine. Bilirubin produces the characteristic yellow-brown color of feces.

Hemoglobin molecules are tetrahedral in structure, consisting of four heme groups surrounding a globin group. Heme, which accounts for only 4 percent of the weight of the molecule, contains all the iron and gives a red color to the molecule. Globin consists of two linked pairs of polypeptide chains. The development of each chain is controlled at a separate genetic locus. The amino acid sequences in these chains have been fully worked out; single or multiple substitutions along the chains result in abnormal hemoglobin molecules.

The study of abnormal hemoglobin has provided a considerable amount of information on human evolution and history. Hemoglobin S, for example, is found in cases of sickle-cell anemia, a severe, hereditary form of anemia in which the cells become crescent-shaped when oxygen is lacking. The sickling trait is found almost exclusively within people of African descent. Other hemoglobin variants occur in various parts of the world and help scholars to trace past human migrations and to study genetic relationships among contiguous and separated populations.

Molecule Visualization

There are several software packages that are useful in visualizing a customized representation of proteins, including Rasmol/Chime, Swiss-PDB Viewer (DeepView), VMD, Pymol, UCSF Chimera, Cn3D, and Jmol. We will first use Jmol and Cn3D and then will create our representation with VMD.

Step 1: JMOL

Browse the PDB web site (http://www.rcsb.org/pdb/home/home.do) and search for hemoglobin. There are numerous results (typically 700+). Some of the search results include 2GTL, 1HV4, 1SI4, 1NGK, etc. Choose one of the results by clicking on it. Next, select the “3D View” tab.

Use your mouse to drag, rotate, zoom in and out, and to identify atoms and bonds of the structure. With JavaScript enabled, you should be able to rotate and scale the image.

Figure 1: PDB Protein View

Now, search for “deoxy human hemoglobin” (Organism: Homo sapiens) and read the abstract regarding 1HV4.

Look up for the molecule called “1GCV DEOXY FORM HEMOGLOBIN FROM MUSTELUS GRISEUS.” To obtain this, search for “deoxy human hemoglobin 1GCV.” Read the abstract regarding 1GCV and differences between human and fish hemoglobin.

Take full advantage of the website. Notice the header information on the molecule, comments/remarks, residue count, the listing of all 4722 atoms, the element, position, angles between atoms, etc. From the “Structure Summary” tab, view the structure in 3D as provided by JMOL.

Also see: http://www.nlm.nih.gov/medlineplus/ency/imagepages/19510.htm

Download and save 1GCV structure (Fig. 2) in PDB format.

NOTE: There have been several issues regarding downloads via the Blade this semester. You may need to do the download portion BEFORE logging onto the Blade.

Figure 2: Downloading PDB File

You may also get the protein sequence by clicking on the “sequence” tab and then clicking on “FASTA” to download/open the amino acid sequence. Unzip the downloaded file using 7-zip (right click-> 7-zip-> extract).

Save for later.

Step 2: Cn3D

In a new tab, search for the same molecule (“deoxy human hemoglobin 1GCV”) in the NCBI structure database (https://www.ncbi.nlm.nih.gov/search/). Then scroll down and select “Structure”.

Figure 3: Selecting structure

You should be able to view the structure in the viewer. Double-click on “Full feature Cn3D Viewer” and explore the different options.

Go back to the previous page.

Next, download the structure data in Cn3D format, as shown below. It should be already prepared for you.

Figure 4: Downloading Cn3D Format

NOTE: Again, there have been several issues regarding downloads via the Blade this semester. You may need to do the download portion BEFORE logging onto the Blade.

If you go this route, save the file in a drive that you can access from the Blade.

Open the downloaded file via the Cn3D viewer available on the Blade server.

Step 3: VMD

VMD provides a variety of methods for rendering and coloring molecules and is freely available for use. The software is useful for visualizing enzymes with wireframes, ball and sticks, and ribbons. From these basic visualizations, colors and beads can be added to highlight certain parts of the 3D structure. It can also animate and analyze the trajectory of molecular dynamics simulations. There are no limits on the number of molecules, atoms, amino acid residues, or animations used within VMD.

VMD is offered on the Blade. To access VMD, open up a file viewer and go to this PC → ULayeredImage(C:) → Program Files (x86) → University of Illinois → VMD → vmd

Figure 5: Opening the VMD Application on the BLADE

After opening the application, it should load the VMD interface and open in. This will look like the image pictured in Figure 6.

Load a molecule

In the VMD Main window, click “File” and then “New Molecule.” A new browser window will open asking you to select a filename. Go to downloads and select your 1gcv PDB file, making sure the file type is set to PDB. Click “load”. You can now close the molecule file browser window, as we will only work with one protein in this lab.

The VMD Main window has loaded a new molecule, ID=0, that contains 4722 atoms. The display window should show the molecule, along with all individual atoms properly organized.

Figure 8: Visualized Protein

Figure 7: Main Window View

You can interact with the 3D display. Initially, your mouse is set to rotate the display. You can click inside the display window and (while holding down the mouse button) rotate the view along the x, y, and z-axis by moving your mouse. Press the “s” key to switch your

mouse to scale mode. Now you can resize the display by clicking and dragging the molecule left and right. Press the “t” key to switch your mouse to translate mode. Now you can translate/shift the display by clicking and dragging the molecule left and right. Press “r” to return to rotate mode.

Change the representation of a molecule

In the VMD Main window, click “Graphics” and then “Representations.” This window lets you change the color and representation of the entire molecule or arbitrary sets of atoms or amino acids (residues). By default, you are given one representation using lines, coloring by name, and displaying all residues. Explore different options. Change the “Drawing Method” → “Bonds”, “CPK”, “Licorice”, “Trace”, “Tube”, “Ribbons”, “NewRibbons”, “Cartoon”, “NewCartoon”, and “Beads”. Notice the differences.

“Cartoon/NewCartoon” is often useful as it displays the alpha helixes in cylinders/spirals and beta sheets using directed arrows. “Beads” represent each amino acid residue based on size and is colored accordingly; this is also useful for highlighting certain parts of the structure but often do a poor job of visualizing the interior of the whole molecule.

Q1. How many helixes does 1GCV contain? Are there any sheets?

Hint: You should be able to see (or not see - wink wink) how many sheets there are just by looking at the visual representation. If you are having difficulty determining how many helixes there are, you may want to refer to the readings.

Add detailed representations of part of a molecule

Set the representation to “NewCartoon” and rotate the molecule, so it looks like a barrel or donut. Create a new (second) representation by clicking on “Create Rep”, set to a default of “NewCartoon” – name - all. Now we will use the second representation to highlight small sections of the molecule and display the subset as beads. Click the “selections” tab and delete “All” in the “selected atoms” box; this box is where you specify which amino acids to visualize in the currently selected representation. The bottom half of this window is a wizard for entering in which residues to display. In the “single words” box, scroll down and double click “heme” to automatically add “resname HEM HEME” to the selected atoms box. Click back on the “draw style” tab and change the drawing method to “beads”. Note how the four hemes in hemoglobin are now highlighted in the molecule.

Figure 9: Creating a NewCartoon

Change the second representation to highlight all of your favorite amino acids. In the “selections” tab, delete “resname HEM HEME.” In the “keyword” drop box, select “resname” (residue name), and in the right drop box (“value”), select your favorite amino acids (e.g., alanine, tryptophan, etc.). Alternatively, you could select the hydrophobic residues, charged residues, first 400 residues, an arbitrary set based on position, etc. Click “apply” to display these amino acids.

Figure 9: Changing Drawing Method

Figure 9: Creating a NewCartoon

Figure 10: Selecting Various Amino Acids

Going further

· Search the PDB website for “COVID-19.” Note the structure, 6Y84. Read more about 6Y84 at https://www.ncbi.nlm.nih.gov/Structure/pdb/6Y84.

· Download the PDB file and visualize the complete structure using “NewCartoon.”

Figure 11: Displaying visualization of COVID-19 structure using NewCartoon style.

Q2: How is structure of toxic protein synthesized by COVID-19 different from Hemoglobin?

· COVID-19 (2019-nCoV, SARS-CoV-2) is the infectious disease caused by the most recently discovered coronavirus. This new virus and disease were unknown before the outbreak began in Wuhan, China, in December 2019 (WHO). More is yet to be understood about the 2019-nCoV. People with underlying medical conditions are considered at higher risk of contracting the severe disease. In real time, we can join in the investigation of emerging pathogens (i.e., zoonotic diseases) that are transmitted from animals to humans (e.g., the coronavirus), https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7128549.

Q3: How might 3D p rotein visualizations help lead to COVID-19 eradication?

· Groups of proteins often have similar functions due to analogous shapes. If you find the topic interesting, you might find other proteins with barrels, ladders, lattices, and structures useful to penetrate membranes, and other notable secondary structures/functions. You could use the “Extensions” menu in the VMD Main window to create animations and simulations of protein-protein interactions, protein-h2o interaction, and protein-ligand interaction.

Future Work (Optional Steps)

· Find additional proteins similar to COVID19-6Y84.

· Use the color option under “draw style” to differentiate between multiple representations. As an example, highlight hydrophobic residues as chrome colored beads and the starting 50 residues in a molecule using a ghost color.

· Find similar protein sequences with high similarities (>70% identities) and load multiple sequences into VMD. Overlay the molecules. Alternatively, find a protein and ligand that are known to interact. Note that you can fix one molecule while still being able to rotate the other(s) by toggling the red/black “F” option for each molecule listed in the VMD Main window.

Self-Reflection: Do these other protein sequences with high similarities have similar functions to the coronavirus? (E.g., are they also viruses?) Are there other coronaviruses?

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