Many of these questions can be answered by referring to the images at http://www.umass.edu/microbio/chime/antibody/index.htm. It will also be helpful to refer to the appropriate chapter of a textbook of immunology.

These questions will help you focus your tour of antibody structure. It is recommended that you print this page and write your answers. Before leaving class today, have your answers checked by your instructor.

For those interested in learning more about protein structure visualization, Protein Explorer is easy-to-use freeware you can use on your computer. You can look at cytokines, MHC, TCR, CD4, HIV gp120, etc. Instructions, with links to those molecules, are at the end of this document.

Ig Domain

1. How many beta sheets are there in an Ig domain? How many beta strands are there in each sheet?

2. Are adjacent beta strands (in the sheets) parallel or antiparallel? (In the cartoon representation, the arrow begins at the amino N-terminus of the strand, and points to the carboxy C-terminus [arrowhead].)

3. What two types of bonds are largely responsible for holding the two sheets together?

   Fab:Lysozyme

4. What kinds of bonds hold the heavy chain fragment to the light chain fragment in the Fab?

5. What kinds of bonds hold the antigen (lysozyme) to the antibody?

6. How many CDR's are there in the paratope? (The paratope is the site on Ab to which the Ag epitope binds.)

7. Draw a map of the paratope showing the relative positions of the CDR's.

8. Which CDR's are most variable? Where do these CDR's lie in the paratope?

9. How many of the epitope-contact atoms in the paratope are not in the CDR's?

10. How many amino acids of lysozyme are in contact with the Fab? How many separate blocks of sequential amino acids are in the epitope?

11. Do all (or most) of the CDR's contact the epitope?

12. There appears to be a hole in the middle of the spacefilled Fab. In reality, what would fill this hole?

    F(ab')₂

13. What holds together the two arms of F(ab')₂?

14. Are the two light chains covalently linked to each other with disulfide bonds? The two heavy chains?

15. What are the light chains covalently linked to?

16. Where are the majority of disulfide bonds in IgG, and what do they link together?

    Whole IgG

17. How many polypeptide chains make up an IgG molecule?

18. Which domains have carbohydrate attached? How might this carbohydrate contribute to the function of these domains?

19. What is the function of the thinnest part of the IgG molecule?

20. What important feature of antibody function is not visible in these images?

21. What kind of chemical bond does pepsin break when it makes F(ab')₂?

    Comparisons between moieties

22. How many immunoglobulin domains are there:

    1. In IgG?

    2. In F(ab')₂?

    3. In Fc?

    4. In Fab?

    5. In Fv?

23. How many variable domains are there in each of the above structures?

24. In IgG, how many pairs of Ig domains are there? How many of these pairs are not directly attached to each other, side to side? Why aren't they?

25. SDS PAGE separates proteins according to their molecular weights. The molecular weight of IgG is 150,000 Daltons. What is the molecular weight of:
    1. a single immunoglobulin domain?
    2. Fab?
    3. F(ab'₂)?
    4. Fc?

26. Can Fc be clearly resolved from Fab by SDS PAGE?

27. What are the approximate dimensions of a single Ig domain? (Hint: use the distance reporting mode available from the utility menu. It reports in Angstroms.) What about the nose-to-tail distance for a whole IgG molecule?

28. What is the approximate size of a protein epitope? (Measure the largest distance between contact atoms in the CDR's.)

29. A lymphocyte is about 10 micrometers in diameter. How many Angstroms is this? What is the approximate ratio of the diameter of a B lymphocyte to the diameter of its Ig receptor?

End of questions.

1. Protein Explorer is freeware that makes it easy for you to look at the structure of any protein for which a structure has been published. Protein structures cannot be accurately predicted from amino acid sequence. They are determined experimentally by X-ray crystallography, or sometimes by nuclear magnetic resonance (NMR). Structures have been published for several thousand different proteins, and many mutated proteins. All published macromolecular structures are available from the Protein Data Bank (PDB).

2. If you go to this page on the web, you can click on the following hyperlinks. Go to www.umass.edu/microbio/chime/antibody/abquests.htm

3. The Fab:lysozyme structure used in the above tutorial is 1FDL. The FirstView page in Protein Explorer explains the view of 1FDL it shows you initially. Then, click on Explore More, and you will go to the QuickViews menu page, where you can try these:
   • To see the secondary structure,
     SELECT Protein, DISPLAY Cartoon, COLOR Structure.
   • To see the hydrophobic cores,
     SELECT Protein, DISPLAY Spacefill, COLOR Polarity2.
     Now press the [Slab] button.
   • To see the antibody atoms (in the paratope) that contact the lysozyme epitope,
     SELECT Chain Y, DISPLAY Contacts.
     Read the help in the middle frame to understand this image.
     Click the [Center] button, then Cancel.
     Click the [Zoom+] button about six times.
     When you no longer want the surface, DISPLAY *Hide*.

   • To see CDR3 of the heavy chain:
     1. If the surface is still showing, DISPLAY *Hide*.
     2. Click on the molecule information button (red and yellow, near the Center button).
     3. Click on Seq3D.
     4. Click on [Show all as backbones].
     5. Select Show range.
     6. Touch the protein sequence in the bottom frame with your mouse, and notice how the amino acid 3-letter code and sequence number are displayed in the slot above. In chain H, find Glu 98 and click it. Then click Tyr 105. You should now have 79 atoms selected (look below the molecule for a count).
     7. Look at where CDR3 of chain H is positioned in the complex.
     8. Enter "define cdr3 selected" in the command slot.
     9. SELECT Protein, DISPLAY Spacefill, and look.
     10. SELECT Chain Y, DISPLAY Contacts.
     11. Enter the command "select cdr3". (It should select 79 atoms.)
     12. COLOR Green.
     13. Observe which atoms of CDR3:H contact the epitope (balls).
     14. Observe the central position of CDR3:H in the paratope.

   • The CDR's in 1FDL are:
     • CDR1:L 24-34
     • CDR2:L 50-56
     • CDR3:L 89-97
     • CDR1:H 31-35
     • CDR2:H 50-65
     • CDR3:H 98-105

4. Other molecules of interest:
   • Tetrapeptide Lys Ala Ile Thr (KAIT) (4-7:B from 1ROG).
   • Immunoglobulin domain (light chain constant domain, 105-214:L from 1FDL).
   • F(ab')₂ (modeled using a symmetry operation on 2IG2, the structure of Fab from a human IgG1, from myeloma patient KOL).
   • Intact IgG1 (modeled from 2IG2 plus 1FC2 and kindly provided by Eduardo A. Padlan of the National Institutes of Health).

   • Miscellaneous molecules including hemoglobin, lipid bilayers, the rhinovirus capsid, ATP, olestra, NaCl, etc.

   • MHC Class I, mouse, presenting a peptide from sendai virus nucleoprotein (2VAB).
   • MHC Class II, human HLA DR1, presenting a peptide from influenza virus hemagglutinin (1DLH).
   • T Cell Receptor, complexed to HTLV1 viral peptide presented by HLA-A2 (1AO7).

   • Interferon Gamma, bovine, expressed in E. coli (1D9C).
   • Interleukin-4 complexed to alpha-chain of receptor, human, expressed in E. coli (1IAR).
   • Interleukin-1 receptor complexed to IL-1 receptor antagonist, human, expressed in E. coli (1IRA).
   • Chemokine IL-8, human (mutated), expressed in E. coli (1ICW).
   • CD4 (human) complexed to HIV gp120 and Fab (human) (1G9M).

5. Want to look at something besides antibody? The easiest place to find a protein structure, using the name of the protein, is at PDB Lite (www.pdblite.org).

6. Once you find a protein, note its 4-character PDB identification code. Then go to Protein Explorer (www.proteinexplorer.org) and enter the code in the slot at the FrontDoor.