VILLIN MODEL CONSTRUCTION
INTRODUCTION:
The construction of this model could have been made out of two types of media.
Wood was considered, but rejected, due to its fragile nature. Metal as a
construction media will make the model stronger, but will make construction
complex. Early on it was decided to allow one aluminum sphere to represent an
amino acid. Titanium-steel alloy tubing would connect the spheres. This alloy is not
extremely expensive and yet provides a durable and light type of connector. The
metal model will be light and have the required strength.
ROAD MAP:
The villin model consists of 14 different amino acids, that is 36 amino acids long. By
protein standards this is a short amino acid sequence.
The three dimensional coordinates were available from Dr. Pandee. Using
trigonometry, these coordinates were changed into two critical angles ( θ, ψ ) and a
length ( r ), which represented the bond length.
θ = tan –1 ( y / x )
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ψ = tan –1 ( z / ( x2 + y2 ) –1/2 )
r = ( x2 + y2 + z2 ) –1/2
Gary Benz
The protein was visualized from six different perspectives. The CHIME program
was used for this purpose. The protein was pictured from the two ends of the three
axes, x, y, and z. The copies were made with each amino acid color coded and the
model form used a backbone visualization mode. These pictures were made,
because construction of the aluminum spheres holes were not very accurate and
some problems did occur during assembly. The exact bond length and angle could
be determined, nevertheless guarantying these dimensions was not generally
possible. These visualizations kept the model accurate. Any gross errors that began
to creep into the model could be fixed using the visualizations.
CONSTRUCTION OF PARTS:
Each amino acid is composed of two different parts. The alloy tubing was cut to a
specified length ( r ) using a metal saw. Each cut was sanded to provide a good fit.
Each one inch diameter aluminum sphere had two holes drilled into them. Two
marks were made on each sphere. The angle in between the dots was based on the
angle ψ. The visualizations are important, because here is where the greatest error
occurs. The hole angle is only accurate to ± 3o. Each hole was drilled to a depth of
1
3
inch and a diameter slightly less than 16
inch. The holes are again drilled with a
4
3
16
inch reamer. The use of a reamer makes a tight fit between the sphere and the
alloy tubing. Duplicates of each sphere are made, because I quickly realized that the
use of a reamer in novice hands, does not guarantee a tight fit. This drilling was
monotonomous and was also a source of error.
35 pre-cut pieces of alloy tubing and 72 drilled aluminum spheres were cleaned first
soap and water. Grease and oil were removed by soaking the materials in a bath of
acetone and then they were allowed to dry.
One or two drops of loctite glue were placed inside one of the metal holes of a
sphere. The alloy tubing was then twisted into the aluminum sphere. Half of the
time the tubing did not have a tight fit. When this occurred the duplicate sphere
was used. If neither worked well, the connection was made by hammering the
sphere into the alloy connector. The aluminum sphere must be protected from the
hammer by using a small cloth towel. The glue was allowed to dry for 24 hours.
Finally, each amino acid was color coded and painted. The alloy tube end was taped
with a quarter inch piece of scotch tape. This kept the end from being painted. In
order for the loctite glue to bond the amino acid pieces together, the reciprocating
pieces must be clean metal surfaces.
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Gary Benz
1.
Phe
BLUE
2.
Leu
RED
3.
5.
7.
Gly
Glu
Lys
GOLD
ORANGE
GREEN
4.
6.
Lys
Lys
GREEN
GREEN
8.
10.
12.
14.
16.
18.
20.
22.
24.
26.
28.
30.
32.
34.
36.
Leu
Gln
Lys
Leu
Leu
Ala
Ala
Arg
Met
Phe
Ala
Phe
Glu
Ser
Met
RED
ORANGE
GREEN
RED
RED
GREY
GREY
DK. GREEN
YELLOW
BLUE
GREY
BLUE
ORANGE
BLACK
YELLOW
9.
11.
13.
15.
17.
19.
21.
23.
25.
27.
29.
31.
33.
35.
Asn
Gln
Trp
Pro
Asn
Phe
Ser
Thr
Gly
Val
Lys
Asp
Asp
Leu
PURPLE
ORANGE
LT. ORANGE
PINK
PURPLE
BLUE
BLACK
BRICK RED
GOLD
RED
GREEN
WHITE
WHITE
RED
The model is intended to show how the tertiary structure of villin is critical to its
function. Other than making the villin model, two hinges will replace the peptide
bond between amino acids. One hinge will occur between amino acids two and
three, while the other will occur between amino acids seven and eight. The hinges
also need to be made of aluminum. This unfortunately introduced a degree of
complexity that could only be addressed by an engineer with metal construction
experience. The hinges would have to be constructed by an engineer.
Free rotation around the peptide bond would be more realistic, but would cause two
problems. First, the model would collide with other parts of the villin model during
rotation. Second, free rotation would introduce a spectrum of possible structures.
Keep in mind only one structure will allow the villin model to function properly.
Instead it was decided that two magnets placed inside two aluminum discs would
eliminate both of these problems. By placing the magnets 90o apart from each
other, only two possibilities would exist. By having two hinges ( the discs
sandwiched together ) only four possible structures exist. The diagrams of the
hinges are attached as an appendix.
ASSEMBLY:
Assembly will occur in three phases. The villin model will constructed. It will be
made such that it consists of three pieces. Once the pieces are made they will be
connected using the hinges. The final model will be fitted with magnets that will
bind to a constructed receptor site.
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Gary Benz
The villin model is constructed of in three pieces. The first piece consists of amino
acids 1 – 2. The second piece is amino acids 3 –7. The final piece is amino acids 8 –
36. The larger piece presented several problems. The final position that individual
amino acid should take had to be marked off with a large brick. This was needed
because the glue began to dry quickly in the loctite position. The twisting motion
needed to put the amino acids together was a coarse motion, but it had to stop at
predetermined angle. The large brick was set up so I would stop twisting when the
amino acid reached the brick, which represented the correct angle. When the
amino acid sequence becomes longer it had to be clamped down so it would not
move during the twisting action.
Often the pieces would not loctite together and this required the use of a hammer to
pound the alloy tube into the reciprocating aluminum sphere. Even when the
sequence was clamped down, hammering sometimes lead to the sequence breaking
at a weak point along the sequence. The third piece required a considerable amount
of patience. The loctite glue must dry for 24 hours. I found that only one amino
acid could be added to the sequence each day. This made the assembly a slow
process that took an entire month.
The model is connected by replacing the alloy tube with the hinges. This was the
cause of the model not being finished by the end of November 2003. The hinge did
not have the right magnet strength, despite trying to shield the magnet or moving it
deeper into the individual discs of the hinge. These hinges are designed to be
attached by connecting to two small alloy tubing originating from each adjacent
amino acid. Each disc of the hinge has a hole drilled into it to receive the alloy
tubing. The mass of these hinges may require some type of model support in
between amino acids 9 and 10.
The villin model can then be placed in the one out of four “correct” positions. The
receptor site for the villin model is represented by a movable dowel, which will have
magnets mounted on them. One magnet is place on either amino acid 1 or 2.
Another is placed on one amino acid in the sequence 3 – 7, and finally a magnet is
placed on either amino acid 8 or 9. The magnets should be placed so that if
connected with imaginary segments, the resulting line would be as vertical as
possible.
The receptor site will have three magnets placed on it so it will bind to the magnets
on the villin model will bind to it.
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Gary Benz