Project

Our project modeled the interaction between a glucose molecule and a previously engineered and tested glucose biosensor (Fig. 1).

Figure 1 - Our biosensor in its environment. The protein, colored yellow and in the drawing method New Cartoon, is in an aqueous environment, which is shown by the blue molecules. Glucose is bound to the biosensor, and is colored red.

We modeled the biosensor in the presence and absence of its target, glucose (Fig. 2, 3). The biosensor we researched contains a periplasmic binding proteins and protein fluorophores. These proteins and other proteins are made up of long chains containing a specific sequence of amino acids. There are 21 core amino acids within the human body, each containing a carboxyl group, amino group, and an R-group all bonded to a central carbon atom. The R-groups are what determine the identity of the amino acid, and it is made up of different atoms.

Atoms are the smallest unit of matter from which most of the world, including our biosensor, is built. Three properties largely contribute to the behavior of atoms: charge, sterics, and free energy. In a neutral atom, the number of positively-charged protons and negatively-charged electrons are equal, resulting in a zero net-charge. However, the removal or addition of electrons throws this balance off, creating a positively or negatively charged atom. Similar to magnets, positive atoms and negative atoms will attract each other, while atoms of the same charge repel. Sterics involves the spatial arrangement of atoms. When atoms get too close to each other, their electron clouds repel each other, creating an unstable molecule. Finally, Gibbs free energy measures the ability to do work. Atoms try to move towards a lower energy potential, and lower their ability to do work. All three of these factors affect the behavior of atoms, and therefore the properties of proteins.

Figure 2 - Our biosensor without a glucose molecule. The biosensor is colored yellow (representation: NewCartoon).

Figure 3 - Our biosensor bound to a glucose molecule. The biosensor is colored yellow (representation: NewCartoon) and the glucose molecule is colored blue and red (representation: VDW).

Our biosensor requires the presence of glucose to induce a change in the periplasmic binding protein (Fig. 4, 5). This change in shape allows the protein fluorophore to illuminate and provide a visual indication of the presence of glucose. The proteins are illuminated using FRET, or fluorescence resonance energy transfer. FRET allows one protein to transfer energy so another protein, which then fluoresces. This procedure is dependent on distance, therefore the change in shape is necessary to bring proteins close enough together to allow for illumination.

Figure 4 - This is the catalytic domain of our original biosensor without glucose bound to it.

Figure 5 - This is the catalytic domain of our original biosensor with glucose.

Our team used the program Amber to produce the input files for our simulations, specifically the program LEaP, a branch within Amber. The pdb file containing our biosensor was first loaded into the program. We then edited the molecule’s environment by adding 113 ions to the aqueous environment. LEaP was also used to bond a glucose molecule to our biosensor, thus creating two varying environments, one with glucose present, and the other without, and allowed us to run two simulations and compare the results. The edits made to the biosensor and its environment were saved as prmtop and inpcrd files. The prmtop file contained the topology of the model, while the inpcrd file contained the coordinates. These files were then loaded into NAMD for the simulation.

NAMD was an essential part of our process, as it allowed us to load the environments we had created and the parameters we had set in AMBER, and test our biosensor’s functionality in each of the 12 environments we modeled.

We used VMD to provide us with a visual representation of our final project. Most VMD default parameters were used, however when representing building the biosensor we used a different drawing method called CPK. This allowed us to see every bonded atom making up the biosensor. Different representations allow for us to see different aspects of the biosensor. For example, using the representation New Cartoon allows one to see the molecule’s alpha helices and beta sheets, not each individual atom.

VMD was a useful tool in our project because not only does it represent standard molecules read from PDB (Protein Data Bank) files, but with its integration with NAMD, it could represent more complex systems; a large protein or molecule in a specified environment. VMD was not used for applying any parameters on either the system or the molecule itself; it was only used to created a 3D model of the system.

Using these three programs -- AMBER, NAMD, and VMD -- we were able to not only build our system, but model the biosensor in it, adjust the environment to a realistic one modeling the blood stream, and visualize our biosensor.