Using the Protein Data Bank to Examine the T4 Lysozyme Structure

Open Internet Explorer

1. Type the URL: http://www.rcsb.org/pdb/   into the address section of your Internet browser. This brings you to the Protein Data Bank (PDB).

2. In the Search section type 257L. Press Enter.

3. This brings you to the data bank for the T4 lysozyme protein we will be working with this semester. Note that this protein is a mutant version of the wild-type version of T4 lysozyme. It has a metal-binding site engineered into the protein in order to facilitate protein purification. Also, it has an amino acid change at residue 54 (Cys replaced by Thr) and at residue 97 (Cys replaced by Ala). These changes increase the stability of the protein. The 3-D structure of this protein varies very little from the wild type. You can convince yourself of this by repeating today’s exercise with the wild-type structure (2LZM instead of 257L).

4. Click on Sequence. Answer these questions (Include the answers as part of your assignment below):

    a. How many subunits does the enzyme have?
    b. How many amino acids are in the protein?
    c. How many alpha helices are present?
    d. How many beta strands are present?

5.Go to the bookmarks on your computer and open the homepage for the NCBI (National Center for Biotechnology Information). Alternatively, you can type in the URL http://www.NCBI.nlm.nih.gov
Type 257L in the search. On the menu that comes up, scroll down to Structure: 3-dimensional macromolecular structures. Click.

6. This will bring up another screen.

7. Click on the picture of the crystal strucure. From the larger picture that pops up click on view 3D structure. In the box that pops up, click on save. Save the file in Desktop so you can easily find it later. Note its name (nmdbrsv) so you can select it later.Your particular computer may already have this structure saved in Desktop from a previous student or class. If so, replace that file with yours. Exit Internet Explorer. Go to My Computer. Click on hard disc C. Then click Program Files. Choose to show the files, ignoring the warning. Click the NCBI link. Then click Cn3D4.1. Then click on Cn3D. The symbol and controls will pop up on your screen. Go to file on the pop-up menu. Click Open. Then, using the look in line, open Desktop and select the nmdbrsv structure of your protein that you previously saved in the Desktop. Click open. The interactive structure and associated sequence and commands should pop up on your screen. You can now manipulate your structure. Use the menu to view the structure in a variety of ways. You can move the structure around with the mouse to view different angles. Look below the structure picture and you will see the amino acid sequence. Find the threonine at residue 54. Click it with your mouse arrow. Notice it turns yellow. Now look at the structure picture above. Do you see the location of the threonine there, highlighted now in yellow? That is the site of the substitution in this protein mentioned above. Many other applications can be done here as well. To learn more about how to use this program, type this URL http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3dtut.html
You are not required to learn this program but, if you are interested, feel free to explore it and apply it to our T4 lysozyme protein. You should also repeat this entire exercise with the wild-type structure by using 2LZM in the structure search. Include any interesting differences you find between these two structures and also compare what you find using the NCBI site with what you found in the PDB site. Note any differences and discuss them as part of your summary report.

This semester you will also be using another expression vector that makes a protein that has been crystallized. This is the CIB protein (calcium binding protein) currently under study in the laboratory of Dr. Uhlas Naik in our department. The PDB number for this crystal structure is 1Y1A. Repeat the above exercises that you did for the T4 lysozyme protein structures with this CIB protein. Answer these questions:
    a. How many chains does the protein have?
    b. How many amino acids are in each chain?
    c. How many alpha helices are in each chain?
    d. How many beta strands are in each chain?

Assignment: Find information about T4 lysozyme and write a 5-6 page summary of what you have learned about this protein and its biological role. Also include answers to the questions above in this report. Please include a bibliography of the references you used to generate your report. The summary will be due at our next lab meeting and is worth 5% of your final course grade. Be prepared next week to orally discuss what you have learned from your research about T4 lysozyme with the rest of the class. We will take turns sharing interesting aspects of T4 lysozyme with each other. This will be done very informally. Also include the answers to the CIB protein questions (see above) as an addendum to your T4 report. You will not write a formal paper about the CIB protein.