(1) goto PDB link to download:
                                            1ZNI (porcine insulin)
                                            1LPH (engineered human insulin)

(2) Double click on the Raswin icon on your computer desktop. This should call up the RasMol windows (the RasMol Command Line window will appear minimized).

(3) Click on the minimized RasMol Command Line to display the command line window.

(4) Type: background white, followed by a return. This changes the color of the background from black to white.

(5) You can rearrange and size the windows to suit you.

(6) Drag PDB file - 1ZNI and drop down to the rasmol window. You now should see the structure of insulin displayed as a wireframe model in the main window! (look also for command window.)
 

                  How many polypeptide chains (type the follow commands in the command window)

               reset
               restrict backbone
               ribbons on
               wireframe off
               color chain
 

                  One can also save a script to your floppy disk by typing: write script A:\zni.sp
               (or whichever directory that you want)
 

                How many amino acids in chain A?

                    select *.ca and *a (select all CA atom on chain A)
                        (answer given in the command window: "21 atoms selected!", so there are
                         21 amino acids in chain A)
 

                That is N/C- terminus?

                    select 1,21 and *.ca and *a
                    label
 

                    (9) There are four polypeptide chains in this structure of insulin, each shown with a different
                    color. A PDB file uses a unique letter for each chain, beginning with "A", which RasMol
                    recognizes. For example, try the RasMol command restrict *a. This displays only the "A"
                    chain in the RasMol view window. How could you use RasMol to identify the N- and
                    C-terminal residues of the "A" chain?

                    (by click both ends and see what is in the command window)
 

                   10.Is there more than one type of helix in the "A" chain?
 

                        select 13-16:a and *.ca
                    spacefill 200

                        select 16-19 and *.ca and *.a
                    spacefill 200

                    then check how many amino acid per turn.
 

                   11  What is the secondary structure of chain B?
 

                   12 .How many amino acids are in each chain? Do any of the chains have the same amino acid
                    sequence?  The PDBSum link for 9ins has the answer.

                    http://www/biochem.ucl.ac.uk/bsm/pdbsum/1zni/main.html
                    (Please explore this site further after the class)
 
 

(13) Explore the various pull-down menus to change the presentation of the structure, under Display, Colors, and Options.

(14) Explore the functions of the left mouse button by clicking on the structure, and by dragging the mouse. Try dragging the left mouse button with the shift key held down. Try the same but with the right mouse button held down.

(15) type select HOH. Then display as ball and stick. What do you see? Display as spacefill, then as wireframe. What do you see?
 

(16) Show Those Disulfides

 Now let's extend our discussion. Insulin is secreted by the
 pancreas into the blood stream. Secreted proteins often contain
 disulfide bonds (-S-S-) due to an oxidizing environment. They are
 formed when two cysteine side chains (-CH2SH) come within
 about 0.3 nm (3 Å) of each other. Let's use RasMol to see if the
 "AB" monomer contains any disulfide bonds. We can do a quick
 test of this by using the following commands:

                  reset
              restrict *a,*b
              ssbonds on
              ssbonds 100
              color ssbonds yellow

                                   Each amino acid in a protein chain is numbered in sequence,
                                    beginning with the N-terminal position. Identify the cysteine residues,
                                    by number and chain, that are involved in disulfide bond formation.

(17) You learned that the AB insulin monomer has three
 disulfide bonds. Let's do a better job of displaying them:

               reset
             set bondmode or
            select sidechain and cys and (*a,*b)
              wireframe 70
              spacefill 200
             set specular on
 

 Instead of seeing just S-S bonds, we can now see the entire
 cystine side chain (-C-S-S-C-), without hydrogens, and its link to
 the protein backbone. Two of the helices in the 21 residue A
 chain are crosslinked to the B chain. In addition, the A chain has
 an intramolecular crosslink. The "CD" monomer is structurally
 similar to the AB monomer. There is, however, a significant
 structural difference in the B and D chains, despite identical
 primary structures.

(18) What's holding those monomers together? (1zni vs. 1lph)
 

 The monomer is only part of insulin's story. In the presence of
 various stabilizing ions, such as Zn2+ and Cl-, insulin monomers
 reversibly self-associate into dimers and hexamers.
 The monomer is the active form in the blood stream because it
 can interact with insulin receptor protein. The aggregate forms
 (dimer, hexamer) are inactive but are physically more stable and
 are therefore used in pharmacological preparations for diabetic
 patients.
 (The physical instability of monomers in pharmacological
 preparations is characterized by partial unfolding (loss of tertiary
 structure). The unfolding exposes hydrophobic surfaces that
 induce long insulin fibrils to form. These fibrils do not reform
 monomers and also elicit an immune response - insulin
 antibodies are formed which are potentially dangerous).

 The insulin response time in vivo depends on the rate at which
 hexamers and dimers dissociate into active monomers following
 injection into the blood stream. The stabilizing ions (zinc and
 chloride) dissociate upon injection, which causes the equilibrium
 to shift toward monomers. The rate of dissociation depends on
 the strength of the interaction forces between monomers. These
 forces occur at the interface between the B and D chains of the
 insulin dimer (see figure at right).

         select all
        wireframe off
        ribbon off
        backbone 350
        color chain
        select *.ca or *.cb
        wireframe 100
        select polar and sidechain
        wireframe 100
        color green
        select hydrophobic and sidechain
        wireframe 100
        color blue
        select charged and sidechain
        wireframe 100
        color red
        restrict :b or :d

        Now try to see what is holding B and D chains together.
 

(19)Compare these two images.

                  P28-K29 in the native is now K28-P29 in the mutant

 The in vivo activity of subcutaneously injected insulin is directly related to how fast aggregate preparations dissociate into monomers. The main effect of the mutant has been to weaken the interaction between B and D chains, but to still allow aggregation (dimer and hexamer formation) in the presence  of Zn2+ and Cl-. Note that the proline side chains are oriented toward the BD interface in the native structure (left image) but not in the mutant structure (right image). The mutant eliminates an important hydrophobic contact, which weakens the BD interface. As a result, hexamers and dimers of mutant insulin show an increased rate of dissociation into monomers following subcutaneous injection. This leads to a much faster in vivo response and, thus, a so-called rapid-acting insulin preparation. The mutant form is marketed under the name Humulin or Humalog (Eli Lilly).