The XOR demonstration points to the possibility of using networks of enzymes to create
We have developed a software simulation tool to investigate the interaction of conformational, kinetic (reaction-diffusion), structural, and dynamic (force) interactions of protein networks in three-dimensional space that for the present purposes can be used to
UV-spectrophotometer ( ƛ = 339 nm); analytic scale; adjustable micropipettes (200 μ l, 1 ml); pH meter; timer.
Malate dehydrogenase from porcine heart, as ammonium sulfate suspension (store refrigerated); NAD + (oxidized β -nicotinamide adenine dinucleotide), as free acid (store refrigerated or frozen); l-malic acid, as free acid; MgCl 2 as magnesium chloride hexahydrate (MgCl 2 6H 2 O); MOPS (3-[N-morpholino]propanesulfonic acid); glycine (aminoacetic acid), as free acid; 10 N HCl and 10 N NaOH (for pH adjustment); pure (distilled) H 2 O. (Below, "water" always refers to pure H 2 O.)
Basis Solution for signals (120 mM glycine, 7.5 mM l-malic acid, 1 l): Dissolve 9 g glycine in about 950 ml water. Add 1 g l-malic acid and allow to dissolve while stirring. Adjust to pH 10.5 with 10 N NaOH. Fill with water to a final volume of 1000 ml.
MgCl 2 Solution (4 M MgCl 2 , 50 ml): Dissolve 40.66 g MgCl 2 6H 2 O in 15 ml hot water. Let the solution cool to room temperature. Fill with water to 50 ml.
Signal solutions: Add 5 ml of water to 100 ml of the signal basis solution (1). The resulting solution is used for 0-signals. Add 5 ml of the MgCl 2 solution (2) to 100 ml of the signal basis solution (1). The resulting solution is used for 1-signals.
Enzyme solution (MDH/NAD + , 10 ml): Dissolve 20.93 g MOPS in about 300 ml water, then fill up to 475 ml. Adjust pH to 7.4 with 10 N NaOH. Fill up with water to a final volume of 500 ml. This is the 0.2 M MOPS buffer. Weigh 36 mg of NAD + into a test tube that can hold 10 ml fluid and is wide enough to access with the 1 ml micropipette. Add 10 ml of the 0.2 M MOPS buffer and shake to dissolve the NAD + . Add about 20 μ l malate dehydrogenase suspension and shake. If the response time for the signal processing is found to be too slow, more of the enzyme suspension can be added to the solution.
The volume of the signal solutions and the reaction solution may need to be adjusted for the particular spectrophotometer used. The minimum volume required to cover the beam
The input signal pattern is
Set the spectrophotometer to continuously record absorbance at ƛ = 339 nm.
To start the processing, pipette 0.5 ml of the enzyme solution (4) into the cuvette containing the signal solutions (6). A timer is started and the cuvette content is mixed (e.g., by inverting the sealed cuvette or by stirring when the enzyme solution is added).
Record the progress of the reaction for various combinations of the input signals by repeating steps 6 through 8. Choose a response time that will separate 00 and 11 input patterns from 01 and 10 inputs and determine the threshold level from the corresponding absorbance values.
Signals can now be
The above protocol can serve as a starting point to explore other signaling substances. It is quite robust and could easily be
The basic concept of the simulator is as
The macrocomponents are represented in the simulation space by dodecahedra, each consisting of up to twelve
Figure 1.7: Schematic of interactions supported by the CKSD simulator (for simplicity limited to a three enzyme system). The enzymes (labeled by e 1 , e 2 , and e 3 ) have from one to three states (labeled by the q i ). States represent conformations. Arrows connecting states represent conformational transitions. These are typically influenced by the milieu components (dashed arrows) and also may be influenced by direct interactions between two enzymes (dashed arrow from e 2 to e 1 ). Specific conformational states catalyze milieu
We assume that
has ten conformational states that
Figure 1.8: Conformational transition used to simulate enzyme e 1 . The diagram is not based on any actual enzyme. The
Figure 1.9: Simulated response surface for enzyme e 1 with respect to signaling substances R and S . The product D is used as the output value. The values in the diagram show the actual number of molecules present in the simulation space. The latter contained 200 e 1 enzymes distributed on a 61 × 61 × 21 lattice.
For the second reaction, catalyzed by enzyme e 2 , we take
Here we assume that e 2 has only four conformational states. As shown in the state transition diagram (figure 1.10), the enzyme is sensitive to the same two signaling substances, R and S , as e 1 . The response surface is shown in figure 1.11.
Figure 1.10: Conformational transition diagram for enzyme e 2 . See caption of figure 1.8 for explanation.
Figure 1.11: Simulated response surface for enzyme e 2 . The space contained 300 e 2 enzymes; cf. figure 1.9.
Now suppose that both enzymes are introduced into the reactor. As can be seen from the reaction schemes above,
Figure 1.12: Combined response surface resulting from interaction between enzymes e 1 and e 2 .