CARBOXYPEPTIDASE A - CHEMICAL AND STRUCTURAL PROPERTIES

NB. It may be worth your while to obtain a hard copy of this tutorial exercise to avoid having to switch back and forth between the Browser and RasMol windows. Choose 'print' from the file menu if you have access to a printer. Students working through the exercise in a tutorial session should have a copy with their tutorial handout.

You will have some questions to answer along the way! These will be indicated by the Q/A icon . When you see this icon, single click on it to go to the appropriate questions.

NB. The following exercise is designed to guide you through some of the basic structural properties of Carboxypeptidase A which it shares with many other proteins. The instructions are a guideline only, feel free to experiment! For further information on any of the commands used in this tutorial, consult the online RasMol Manual

Step One - Begin RasMol

Single click on the Rasmol icon (above) to begin the program. The command and graphics windows should come into view. A wireframe image of Carboxypeptidase A will be loaded into the graphics window - be patient as this may take a while (depending on your system). Position the windows so that you can work in them both. You will be entering commands at the Rasmol prompt :

RasMol>

Remember to click the mouse pointer in the command window when you need to type in the commands !

Step Two - Identifying Di-Sulfide Bonds

You have previously seen the primary amino acid sequence and should have identified the cysteine (sulphur containing) amino acids of carboxypeptidase-A. Now, let's identify the position of the sulphur containing amino acids relative to each other. To do this type in the following commands next to the Rasmol prompt in the Rasmol command line window. Pressing the 'enter' or 'return' key executes the command.

Commands :

select sulphur (enter)

This command selects all sulphur atoms in the molecule

colour yellow (enter)

All selected atoms are now yellow.

cpk (enter)

CPK is the space that an atom occupies, so all sulphur
residues are now 'spacefilled' yellow.

Point the cursor at each of the sulphur atoms and click with the mouse button. Look in the command line window and record the number and identity of the amino acids.

Test your knowledge!

cpk off (enter)

Step Three - Hydrophobic vs Polar Residues

Now compare the distribution of the hydrophobic and polar amino acid residues in carboxypeptidase A. Rotate and resize the molecule at any stage to observe the effects of your commands.

Commands :

select all (enter)

This command selects all atoms in the molecule

colour white (enter)

All selected atoms are now white.

select hydrophobic (enter)

All hydrophobic side chains are selected.

cpk (enter)

select polar (enter)

All polar side chains are selected.

colour red (enter)

cpk (enter)

Observe the distribution of polar and nonpolar amino acid residues

Single click on the icon above if you wish to check your work!

Next we can use the slab command to 'slice' through the molecule at various positions from front to back. We first reset the zoom position to normal. With the slab commands we will work our way from the front to the back of the molecule. What do you expect to observe as you move your way through the molecule?

Commands :

reset (enter)

resets the zoom to normal position

slab 20 (enter)

shows a slice one fifth (20%) of the way through the molecule

slab 30 (enter)

slab 40 (enter)

slab 50 (enter)

slab 60 (enter)

slab 70 (enter)

slab 80 (enter)

slab 90 (enter)

slab 100 (enter)

Test your knowldege!

Step Three - Chemical Properties

RasMol defines 'Sets' of amino (and nucleic) acids based on their structural and/or chemical properties. Predefined Sets are available. For a complete listing of Predefined Sets, maximise the command line window and enter :

help sets (enter)

Let's have a look at a graphic view of some of these sets.

Commands :

select all (enter)

cpk off (enter)

colour yellow (enter)

select acidic (enter)

Acidic amino acids selected

colour red (enter)

Acidic amino acids are now red

select basic (enter)

colour blue (enter)

select hydrophobic (enter)

colour magenta (enter)

You can also define your own sets :

define newset ser,thr,gln,asn (enter)

select newset (enter)

colour green (enter)

Finally, let's look at the CPK model for the sets we've chosen :

select acidic, basic, hydrophobic, newset (enter)

cpk (enter)

Observe what is in common between the acidic, basic and newset of amino acids

Single click on the icon above if you wish to check your work.

Test your knowldege!

Step Four - Structural Properties

Rasmol defines sets for the major structural characteristics of proteins. You can readily isolate the secondary structures in the enzyme using the select command.

In the following exercise, you should OBSERVE the alpha-helical, beta-sheet and beta-turn content of carboxypeptidase A

Let's first look at the alpha-helices present in carboxypeptidase A.
Commands:

select all (enter)

cpk off (enter)

colour yellow (enter)

select helix (enter)

colour magenta (enter)

ribbon (enter)

A ribbon is woven through the backbone of the helices.

How many alpha helices can you see?

Let's now have a look at the beta-pleated sheets :

select all (enter)

ribbon off (enter)

colour yellow (enter)

select sheet (enter)

colour redorange (enter)

ribbon (enter)

Now find the turns in the molecular structure :

select all (enter)

ribbon off (enter)

colour yellow (enter)

select turns (enter)

colour cyan (enter)

ribbon (enter)

Now put all the structures together :

select all (enter)

colour yellow (enter)

select turns (enter)

colour cyan (enter)

select helix (enter)

colour magenta (enter)

select sheet (enter)

colour redorange (enter)

select all (enter)

ribbon (enter)

Test your knowldege!

Now let's isolate an alpha helix and examine it more closely.

Commands :

select all (enter)

ribbon off (enter)

colour yellow (enter)

restrict 13-32 (enter)

Viewing is now restricted to amino acids 13 to 32 comprising one of the alpha helices.

hbonds (enter)

hydrogen bonds between atoms appear as dotted lines.

select oxygen (enter)

colour red (enter)

select nitrogen (enter)

colour blue (enter)

Resize and reposition the helix on your screen if necessary.

select 13-32 (enter)

ribbon (enter)

Test your knowldege!

Finally, let's isolate the pleated sheet structure and examine it more closely.
Commands :

select all (enter)

ribbon off (enter)

hbonds off (enter)

wireframe (enter)

restrict sheet (enter)

hbonds (enter)

ribbon (enter)

Resize and reposition the sheet on your screen as necessary.

Test your knowldege!

Step Five - End of Tutorial Exercise

Select the command window and enter the quit command.