Team:ASIJ Tokyo/Design
Project Design
Improving the Inhibitor
Protein Design
To improve the efficiency of alpha-amylase inhibition by 0.19 α-amylase inhibitor (0.19 hereafter), we examined two limitations of the inhibitor: dimerization, and the propensity for the 0.19-amylase complex to denature. Since dimerization of 0.19 restricts the orientation and movement of the inhibitor protein, it decreases the probability of the inhibitor properly docking to the alpha-amylase, thus fewer interactions with alpha-amylase occur and lower its efficacy. If dimerization were prevented, the 0.19 to alpha-amylase docking ratio would increase, decreasing the rate at which starch is broken down into glucose, theoretically leading to a lower initial blood glucose spike than in those consuming the non-dimerized 0.19.
To solve the problem of dimerization, we modified the amino acid sequence in 0.19 to make the protein chains more repulsive. Specifically, we looked at the charge and polarity of amino acids to determine which part of the amino acid sequence ought to be modified. Furthermore, using Amino Acid Interaction (INTAA) web server [1], we analyzed the interaction energies between amino acids in the chains of 0.19 and determined which amino acids to modify. Considering the similarities in hydrophobicity and Grantham’s Distance of the amino acids, we changed the 84th amino acid—Arginine to Glutamic Acid (Figure 1). We used the protein mutagenesis tool on PyMOL to confirm the size of the amino acid introduced by a point mutation (Figure 2). Finally, we used ClusPro’s dimer probability calculator to see if our modification would prevent dimerization. Our modification was found to decrease the probability of dimerization by 34%, from 76% to 42% (Figure 3.1 and 3.2).
Figure 1
Figure 2
Arginine to Glutamic Acid change in both chains (shown in white)
Figure 3.1
Figure 3.2
The second issue we hoped to address was the denaturing of the 0.19-amylase complex in the stomach. With high pH, proteins denature and the structure loosens. In addition, the bonds between the enzyme and inhibitor weakens, allowing proteases in the stomach to attach to the gap and break down the complex (Figure A). Once the complex is separated amylase is no longer inhibited, so it can readily convert left-over starch to glucose (until it completely is denatured in the stomach acid).
Figure A
Figure A
To increase the stability of the 0.19-amylase complex to resist denaturation, we modified the amino acid sequence of the binding site between 0.19 and alpha-amylase using the same procedure as above. Specifically, we analyzed the amino acid interactions and made a change in the sequence of 0.19 to decrease the repulsion with the amino acids of alpha-amylase. For this purpose, we made two modifications to increase the accuracy of the experiment (Figure 4). Observing that the amino acid with one of the strongest interaction energies was between Isoleucine 44 on the 0.19 and Asparagine 362 of amylase (4.47kJ/mol), we replaced the 44th amino acid of the 0.19 with Asparagine. We chose Asparagine since, it was polar, and it also had a similar molecular weight and shape when compared with Isoleucine. The modification successfully decreased the repulsive energy to 0.00kJ/mol. Similarly, we reduced the repulsive energy in a separate construct by exchanging Glycine 4 for Glutamic Acid which had repulsive energy of 6.21kJ/mol with Histidine 305 of amylase. Similarities in polarity with Histidine, and similarities in molecular weight, and charge of the Glycine provided the basis for our above modifications to the 0.19 protein. This modification decreased the repulsive energy to -0.01kJ/mol, showing a small amount of attraction. Both mutagenesis was introduced to the 0.19 in silico using PyMOL, cconfirming the size of the new amino acid (Figure 5.1 and Figure 5.2).
Figure 4
Figure 5.1
Isoleucine to Asparagine (shown in pink)
Figure 5.2
Glycine to Glutamic Acid (shown in red)
Parts Design
As mentioned in Project Description, we are doing this project in hopes of developing a viable food supplement such as Miso, which uses yeast to create fermented soybean paste. Our vision of the food supplement is one that incorporates 0.19 α-amylase inhibitor (0.19 hereafter) in the production stage, so we modified Saccharomyces Cerevisiae that will constantly produce the 0.19 during the fermentation process. Although yeast found in Miso is a different strain with high-sodium resistance [2], our modified S. Cerevisiae will serve as a proof of concept, demonstrating the possible application of GMO in the production of a food supplement.
All three constructs (Figure 6) contain a constitutive yeast promoter TDH3, which expresses the 0.19 gene as long as the cell continues to thrive. Saturating the Miso is important since high 0.19 protein concentration will yield better inhibitory activity of α-amylase.
Figure 6
