Difference between revisions of "Part:BBa K5396000"

Revision as of 20:03, 20 September 2024

BaCBM2_RFP_3xMad10

This CBM2 protein is fused with the red fluorescent protein (RFP), which exhibits an excitation maximum at 558 nm and an emission maximum at 583 nm. This fusion enhances the visualization of CBM2. The protein also has three MAD10 peptides [ ], which serve as a magnetic tag that facilitates the purification of the protein through magnetic separation techniques.

This part was used as template to construct BBa_K5396003.

Usage and Biology

This CBM2, or Carbohydrate-Binding Module 2, is a protein sourced from Bacillus anthracis. It belongs to a broader family of carbohydrate-binding modules that are crucial for the degradation of polysaccharides. These modules are important to break down complex carbohydrates, enabling microorganisms to convert them into usable energy sources.

Recent study [ ] has shown that CBM2 has the ability to bind to certain types of plastics, especially those derived exhibiting similar structural features of polysaccharides. This binding ability is largely due to the protein's carbohydrate-binding properties, which facilitate interactions with specific functional groups found on plastic surfaces.

Figure 1. AlphaFold 3 3D simulation of BaCBM2 with miRFP and three Mad10 tags.

Characterization

SEC-MALS

Knowing that the protein 6His-CBM-RFP-3xMAD10 has a 44.86 kDa molecular mass (MM), it is possible to observe three different oligomeric states in the SEC-MALS result shown in Figure 2. With a smaller normalized signal (dRI), it was possible to observe a MM of 180±2 kDa around 32 minutes of elution, which is close to the molecular mass of a 4 units oligomer.

Following on, a second elution was made around 34 minutes with 86.2±0.5 kDa. According to this molecular mass, it is possible to observe a dimer structure of the protein with a higher dRI. Finally, with the highest dRI and a molecular mass of 46.1±0.2 kDa at 37 minutes of elution, it was possible to identify the protein monomer state.

When compared to the other oligomer states, it was notable that this last elution had the lowest standard deviation when compared to the others. As a conclusion, it is possible to confirm that the 6His-CBM-RFP-3xMAD10 construction is most likely to be a single unit protein.

Figure 2. SEC-MALS result of 6His-CBM-RFP-3xMAD10.

Circular Dichroism (CD)

If you want to check the comparison between the BaCBM2 and BARBIE1, see our Resultspage.

Plastic Interaction Analysis

Through secondary structures absorbance analysis, it was determined that the BaCBM2 protein structure remains unchanged in the presence of plastic nanoparticles.

The final experimental evaluation using circular dichroism (CD) involved polystyrene nanoplastics. A 100-nanometer particle was introduced into a solution containing BaCBM2. However, as indicated by computational simulations, no conformational change is anticipated. Since CD primarily assesses absorbance related to secondary structures, significant differences are unlikely to occur during the experiment.

Consequently, as illustrated in Figure 6, when the spectrum of BaCBM2 is plotted alongside the nanoparticle solution spectrum, only minor changes are observed. This contrast may be attributed to the scattering effects of the polystyrene nanoparticles, which can introduce noise into the measurements.

Figure Y. Plastic binding protein BaCBM2 spectra acquisition with 100 nm polystyrene nanoparticles.

Protein Corona Formation

The experiment result is shown in Figure X. Although we used a 100 nanometer polystyrene particle, it is important to note that the equipment has a 30 nm standard deviation, which reflects in the plotted result. In an initial moment shown on Subfigure 1 (a), the BaCBM2 is added and starts to aggregate in the plastic particle, increasing the particle size.

On the other hand, in the final titrations, the protein has aggregated in the whole nanoplastic surface, which makes it stabilize its size, as represented on Subfigure X (b). This effect can also be seen in the following points, since there is no size increase.

It is particularly interesting to note the theoretical and experimental comparison. Knowing that the protein has a 2.5 nm width, the expected theoretical particle size was a 5% increase (from 100 nm to 105 nm). On the other hand, the experimental value found for the experiment was an increase tax of 5.05% (from 122.7 to 128.9 nm), showing a great proximity between them.

Figure X. Dynamic Light Scattering of the average particle size in function of the protein titration. On Subfigure (a), it is shown the initial titration state representation and the final state on Subfigure (b).

Sequence and Features