Molecular Models: Amino Acids & Proteins
"Form ever follows function."
-Louis Sullivan (architect)
I. Examine protein primary structure using the CPK model kit
The CPK Model Kit
We will be using a model kit called the CPK model to build the structure of the amino acid, alanine. The atoms of the kit are color-coded:
black = carbon red = oxygen
white = hydrogen
Each set should contain: 1 C-carboxyl (trigonal) atom, 2 C-tetrahedral atoms, 8 hydrogen atoms, 1 N-amide (trigonal) atoms, 1 single-bond oxygen atoms (two-holed), 1 double-bond oxygen atoms (one-holed), 2 peptide linkers, and at least 6 ordinary linkers.
CPK space-filling models incorporate both the covalent and van der Waals radii of atoms into their design. They approximate the true shape of a molecule, and are particularly useful for studying molecular packing. Each element is represented in CPK models by a sphere of radius equal to the van der Waals radius. This is cut perpendicular to the direction of the chemical bonds so that when two atoms are joined by a linker, the correct value of the covalent bond length is obtained.
CPK models are constructed to a scale of 1.25 cm = 0.1 nm = 1 Å.
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The model pieces have different shapes. Although it may
be possible to link the appropriate atoms together using the wrong shaped
pieces, the three-dimensional structure will not be correct and you may
have difficulty seeing the features you are looking for.
The carbon atoms of the carboxyl groups are trigonal and can form bonds three other atoms. One is a double bond to oxygen. Tetrahedral carbons are used in molecules with four constituents bound to the carbon atom. Nitrogen atoms are also trigonal and are used to form the amide (NH2) group of the amino acid. Oxygen atoms are hemispherical (=O) or wedge shaped (-OH). |
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The atoms are connected together by "linkers" that press into the holes in the atoms. Except in special circumstances (more on this later) you will use only the normal (white) linkers. |
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The center carbon atom in an amino acid, called the a carbon, is asymmetric because it has four different substituents attached. This results in an asymmetric molecule in its three dimensional structure. Such molecules are also called "chiral".
Chirality "game" (optional)
Two important classes of molecules containing asymmetric carbon atoms are amino acids and sugars.Amino acids in proteins all fall into one of the two stereoclasses: the L class. (Naturally occurring sugars fall into both L and D stereoclasses.)
These two arrangements (L and D) cannot be converted into each other by rotating or translating the molecules; they are related by the symmetry operation of reflection, as in a mirror (like a left and a right hand).
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| When building a three dimensional model of a molecule containing an asymmetric carbon atom, it is imperative to correctly orient the atoms attached to it. Notice that the COOH and CH3 groups are oriented vertically and go away from you. The NH2 and H groups are oriented horizontally and are coming toward you. |
2. The D isomer of alanine can be made by switching any two groups attached to the chiral carbon. It can also be made accidentally if you have groups coming toward you when they are supposed to be going away or vice versa. Collaborate with a neighboring group to compare L and D alanine (one of your groups should convert your molecule to the other form).
Attempt to superimpose the two molecules by rotating them. Then attempt to superimpose one of the molecules with the image of the other in a hand mirror by rotating them.
3. Convert alanine to glycine by replacing the methyl (CH3)
group with a hydrogen. Now repeat the exercise in step 2. Why
are the results different? (When you are finished, turn both glycines
back into L alanine again.)
The peptide bond has several characteristics that are important in determining the secondary structure of proteins.
The peptide bond formed between the NH of one amino acid and the carboxyl group C=O of the second has a partial double bond character. There is no free rotation around the peptide bond and the C=O and N-H are in the same plane of the molecule.
The other two bonds in the backbone of the polypeptide are able to rotate.
In order to prevent the free rotation of the peptide bond between two amino acids, the gray type of linkers which have a protruding "key" can be used. Some of the holes in the trigonal nitrogen and carbon atoms have slots into which the keys will fit. If you try hard you can force the bond to rotate, so make sure that the H and O atoms are actually opposite each other.
4. During the formation of the peptide bond, a molecule of water is
removed, hence the process is a condensation reaction. Remove an H and
OH as shown in the figures below to form a dipeptide made from your alanine
and your neighbors'.
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The following animation shows the necessary steps.
5. Identify
the carboxyl and amino termini of the dipeptide.
6. When you are finished, make sure that you have taken all of the atoms apart (it is not necessary to remove all of the linkers from their holes) and put a complete set in your bag. Each set should contain: 1 C-carboxyl (trigonal) atom, 2 C-tetrahedral atoms, 8 hydrogen atoms, 1 N-amide (trigonal) atoms, 1 single-bond oxygen atoms, 2 double-bond oxygen atoms, 2 peptide linkers, and at least 6 ordinary linkers.
Build Alanine with a Pushfit Model Kit
Although
space filling models illustrate molecular packing very well, skeletal ("ball
and stick") models
are easier to work with for most other applications.Their
principal advantage is that the positions of the atoms and bonds can easily
be seen. The atoms can therefore
be positioned accurately to correspond to atomic coordinates calculated
from geometric considerations or deduced from x-ray crystallographic analysis.When
a model has been constructed, interatomic distance can easily be measured.
The
scale for the pushfit models is 1 cm = 1 Å.C,
N and O atoms are represented by 0.5 cm diameter balls. H atomic positions
are represented as the ends of short rods coming from the other atoms.
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Special
note: The
most common problem in using these kits is confusion about the piece that
contains the peptide bond (see above). Although this is a single piece, it
is actually part of two adjacent amino acids. It is modeled as a single
piece to prevent rotation around the peptide bond. The practical
implication of this is that when you build a polypeptide, you will always have
parts of incomplete amino acids at both ends of the chain.
1. Construct L alanine with the pushfit model kit. It is important to
assemble carefully so that you do not accidently make the D form.
Here is the piece that represents the rigid peptide bond and which contains part of two amino
acids. Look at the first picture to see which of the atoms represents the
amide nitrogen. You will ignore the part of another amino acid shown on
the lower part of the picture. Attach the chiral carbon as shown in the above picture on the right.
Here is the point at which you can make a big mistake. Notice that in
the diagram, the CH3 group is supposed to go down and away from
you. If you place the dark CH3 model unit either up and away
from you or if you have the chiral carbon with the prongs coming toward
you, you will inadvertently make the D form of alanine rather than the
L form. Make sure that you do this correctly or later structures that you
will build (such as the alpha helix) will not orient themselves
correctly. The model of L alanine is completed by attaching another of the peptide
bond units to the remaining prong on the chiral carbon unit (Note that
it also contains a part of the next amino acid as the first one did):
Build a Polypeptide in Extended
Conformation NOTE: The best strategy in this section is to build a
polypeptide chain as described below and then twist it into the secondary
structures (alpha helix, beta sheet), rather than trying to recreate them from
scratch by looking at the diagrams. 2. Build a polypeptide using all of the pieces in your Pushfit
modeling kit. To do so, just continue the previous steps.
Bond Rotation Varying the angle between adjacent rigid peptide units results
in different protein secondary structures. We will first form our
polypeptide into an extended (linear) form, then modify its shape into
two secondary structures commonly found in proteins. 3. Adjust all alpha carbon angles to put the polypeptide
into extended conformation. This results in an alternation of the
side atoms and R groups in adjacent amino acids as shown below. When viewed from the top, it can be seen that the backbone chain lies
in a single plane. The R groups are offset to alternate sides of
this plane. Build an Alpha Helix 5. Hint: starting with the N terminus at the bottom (as in diagrams), hydrogens
should be pointed down, oxygens up and R groups out. Please note:
because the peptide units contain part of two amino acids fused together,
the last atom on the C terminal end of the chain will be N, and vice versa.
6.
By forming the helix into a tighter or looser spiral, it is possible to form
hydrogen bonds between different oxygen and hydrogen atoms. The correct
"twist" on the helix is one that produces 3.6 residues per turn. Refer to
the left diagram below. Find a red oxygen atom (white ball on your model)
near the N terminus of the polypeptide. Follow the backbone of the chain
until you pass 3 more oxygen atoms. The next nitrogen (white backbone atom
with a white stub on your model) that follows is attached to the hydrogen that
bonds to the oxygen that you started with. Twist your chain around until
each subsequent oxygen atom lines up with each subsequent hydrogen atom. Stryer Biochemistry
4. Begin building the alpha helix by adjusting the bond angles on
the backbone chain so that they look like the diagram below. As this is done, the backbone
will begin to coil into a helical shape, with the oxygens all pointing in one
direction and the amino hydrogens pointing in the opposite direction.
This allows a hydrogen to hydrogen bond to an oxygen from the previous
turn of the helix. Be careful to notice the direction the helix
is twisting.
Computer Models of the Alpha Helix
and Beta Sheet
Click on the following link to open a new tab or window to examine the two
secondary structures.
8.
Alpha helix: 9. Antiparallel beta
sheet: Alternative website: If the site above is down, the questions can also
be answered by visiting A polypeptide in its cellular environment will
spontaneously fold itself into a characteristic three-dimensional structure
that is related to the purpose that it serves in the cell. In addition
to the formation of secondary structures such as alpha helices and beta
sheets, the secondary structures themselves move into particular positions
within the folded polypeptide. The overall shape of the molecule
is called tertiary structure. The positioning of parts of
the polypeptide is influenced by interactions of hydrophilic R groups with
water, attraction of hydrophobic R groups of one part of the polypeptide
with another, and stabilization of the overall structure by disulfide bonds.
In the case of enzymes (catalytic proteins), the
tertiary structure of the molecule produces an active site, a pocket
which holds the substrate in a position favorable for facilitating the
completion of the catalyzed reaction.
Physical Model of Trypsin The notes for this section are included on the problem set page so that
you can take it to the front of the classroom with you when you observe
the TA demonstration. (Questions 1 and 2 are on the problem set page.) 3. Go to the Index of Protein Structures at Molecules To Go, an NIH database.
When you click on the link below, the modeling applet will open in a frame with
these instructions in the left frame. In some versions of Internet
Explorer, the left (text) frame is blank. If that happens, right click on
that frame and select "refresh". Enter "trypsin" as a search term, then press Enter. Click on the
link labeled:
1AND ANIONIC TRYPSIN MUTANT WITH ARG 96 REPLACED BY HI
in the search results. This structure may be fairly far down the list,
and you can use the browser's ""Find" function in the Edit menu
to speed up finding the name. In the "Output requested" drop-down list,
select JMOL PDB viewer. Then click on the "submit
request" button.
4. The initial model is in the ball and stick form. Under "Display
Style" you can drop down a list of other display styles. "Wireframe"
is a view similar to the physical model that you have already observed in the
lab. Change the display style to "Cartoons". Then change the Color
Style to "Secondary Structure" (this drop-down list may have wrapped down to the
next line on your screen. 5. Click and drag to rotate the molecule. Observe the alpha helices
in the molecule.
If you hold the shift key while clicking and dragging you can zoom in or out on
the molecule. 6. Beta sheets are another secondary structure formed when adjacent strands
are held together in a planar arrangement by repeating hydrogen bonds. Observe the beta sheets in the molecule. Are they flat or do
they form barrels? [Note: the individual arrows are not sheets. They
are polypeptide strands that form sheets.] Look at the direction of the arrowheads to see
the antiparallel nature of the sheets.
7. Observe how the ends of the strands in the beta sheets are connected.
8. You can make more sophisticated changes to the display by right-clicking
in the modeling window and making selections from the resulting menus. By
changing the display characteristics appropriately, you can make it possible to
visualize things that would be difficult to notice otherwise.
We would expect hydrophobic side chains to project towards the center of the
protein and away from the aqueous cellular environment around the protein. We
would expect hydrophilic side chains to project outward. So we will change
the display to increase the apparency of side chains expected to be hydrophobic
and hydrophillic.
A. Change the display type to rods. Change the Color
Style to White. Right click and choose: Select, Protein, Polar Residues.
Right click and choose: Color, Atoms, Blue. (These are groups we expect to
be hydrophillic.)
B. Right click and choose: Select, Protein, Nonpolar
Residues. Right click and choose: Color, Atoms, Red. (These are
groups we expect to be hydrophobic.)
C. Now we will highlight the side chains
by getting rid of the backbone. Right click and choose: Select, Protein,
Backbone. Right click and choose: Color, Atoms, Black. Right click
and choose: Color, Atoms, Make translucent.
D. Click and drag the molecule so
that you can examine it from various angles and decide if the orientations of
the two types of side chains are generally as we predicted. You can use a
similar method to examine whether positively and negatively charged side chains
(which are generally hydrophilic) tend to be oriented outwards on the surface of
the molecule.
10. Active site of a trypsin-like enzyme (human
tissue kallikrien 4).
Click on the following link which should open in a new tab or window:
http://biology.kenyon.edu/BMB/Jmol2008/Paige_Anna/index.html In the
Contents section, click on III. Active Site. Skim the text of this section as
you click on the gray squares that highlight the part of the molecule which is
being described. Do not get hung up on the details of how the binding site
works. What you should notice is how the position of particular amino
acids create a pocket that facilitates specific interactions between the enzyme
and its substrate. Several polypeptides (individual molecules) may combine to form a larger
protein. These polypeptides may be the same or different. The
combination of polypeptides in a given protein is called quaternary
structure. For example, normal adult hemoglobin is composed of a
and b subunits acting in concert.
http://www.rpc.msoe.edu/cbm/jmol/1a3n.php (which will open in a new tab or window) 1.
Hemoglobin is a protein found in red blood cells whose quaternary structure is
composed of four globin subunits. Most hemoglobin in adults is composed of
two alpha globins and two beta globins. Click on the Select Alpha-Globins
button then the Select Beta-Globins button to locate the four subunits.
2. Click on the Single Out a Beta Globin button to observe the details of a
single subunit. Click on the Add Heme Group button. The heme group
is an iron-containing group that binds the oxygen carried by the red blood cell.
3. Click on the Color Hydrophobic Amino Acids button. Rotate the
molecule and notice that most of the hydrophobic amino acids are in the interior
of the subunit. 4. Go to the website
http://mcdb-webarchive.mcdb.ucsb.edu/sears/biochemistry/tw-hbn/hbs/hbs1-pdb.htm
. Persons having the genetic disease sickle cell anemia have a mutation
resulting in the change of a single hydrophilic amino acid, glutamate in
position 6 to valine, a hydrophobic amino acid.
A. Click on the HbS1-OFF/ON
check-box to observe the position of this amino acid (colored green). Under conditions of
low oxygen concentration, a hydrophobic patch is exposed at another location on
the protein. Uncheck the HbS1-OFF/ON check-box and check the HbS2-OFF/ON
check-box to observe the hydrophobic patch (shown in yellow). It is
more energetically favorable for the hydrophobic valine-6 on the betaglobin subunit
of one hemoglobin molecule to aggregate with the hydrophobic patch on the
betaglobin subunit of another hemoglobin molecule than to be exposed to
the aqueous environment around the molecule. B. Uncheck both
boxes to see how the two proteins aggregate. Check the beta2-Val6 - Spacefill/Wireframe check-box and rotate the molecule to see the details of how
the two hydrophobic regions interact. Repeated aggregation of many
hemoglobin molecules causes the formation of long chains of hemoglobin molecules
which create fibers that cause the red blood cells to
have an abnormal sickle shape.
http://www.biochem.umd.edu/biochem/kahn/teach_res/Secstruc_jmol/jmol-alphahelix.html
Click on each button at the bottom of the screen in order to see the features of
the helix. You can click and drag to rotate the molecule and look at it
from different angles. In particular, note the role played by the hydrogen
bonds in giving strength to the structure. Also notice how the R groups
are oriented relative to the helix. You can see this most clearly with an
end-on view. While looking end-on, click the spacefill button and see what
happens to the center of the helix.
http://www.biochem.umd.edu/biochem/kahn/teach_res/Secstruc_jmol/jmol-betasheet.html
Click on each button at the bottom of the screen in order to see the features of
the sheet. As with the alpha helix, note the role played by the
hydrogen bonds in giving strength to the structure. Why is this structure
sometimes called a "pleated sheet". Notice how the R groups are oriented
relative to the sheet. In the cartoon view, consider why this is called an
"antiparallel" sheet.
http://higheredbcs.wiley.com/legacy/college/boyer/0471661791/structure/jmol_intro/sec_str.htm
However, the interface is a little less "user friendly".