Our detection module requires coupling with antibodies to achieve detection capability. During the brainstorming and discussions with our teacher, we came up with two ideas. The first one is to use the Streptavidin-biotin system, where Streptavidin is linked to the protein cage component to recognize biotinylated antibodies. The second idea involves connecting the coiled helix of chain A to the protein cage component and chain B to the antibody, completing the construction of the detection module. We are attempting to provide some recommendations for the selection between these two approaches through molecular dynamics simulations.
Introduction
Avidin is a secreted protein obtained from Streptomyces avidinii culture, a type of Streptomyces bacteria. It exists as a tetrameric protein capable of highly specific binding to four biotin molecules. The dissociation constant of the avidin-biotin complex falls within the range of 10^-14 M, indicating an exceptionally strong affinity for biotin.
The coiled-coil, a fundamental structural motif in molecular engineering, possesses a simple yet elegant structure. It consists of two or more alpha helices intricately wound together into a supercoiled rod-like bundle. This organized arrangement is encoded by a seven amino acid repeat denoted as [abcdefg]n. For our study, we selected an artificially optimized coiled-coil with the PDB ID 3HE5.
Structural modeling
To mitigate the potential influence of SpyTag and the linker on the prediction of protein flexibility and stability during molecular dynamics simulations, we opted for GGS as the linker for the fusion proteins in our simulation process. Moreover, during the utilization of ColabFold, we specifically selected and constructed the complex module in conjunction with SpyCatcher to derive both the Streptavidin and coiled-coil components.
Fig. 1. The initial structure of the detection component.(A)Streptavidin component,
Spycatcher(green),SpyTag-Streptavidin (blue).(B)Coiled-coil component,Spycatcher
(yellow),SpyTag-Coiled-coiln(red).
Results of molecular dynamics simulations
Fig. 2. The RMSD of the detection component within 50ns.
Based on the RMSD results, we observed a consistently stable conformation for the Streptavidin component. In contrast, the Coiled-coil component displayed significant fluctuations within a range of approximately 0.35nm for a significant portion of the simulation, indicating a challenge in achieving a stable state for this component.
Fig. 3. The RMSF of the detection component within 50ns.
The Root Mean Square Fluctuation (RMSF) analysis indicates that atoms within the Coiled-coil component exhibit higher flexibility compared to those in the Streptavidin component. Notably, the Coiled-coil region displays pronounced flexibility, suggesting a potential for significant conformational changes prior to binding with another helix. This variability could potentially reduce the affinity between the two helices, affecting their binding interactions.
Fig. 4. The Rg of the detection component.
The gyration radius analysis further supports the stability observation. The gyration radius of the Streptavidin component, being denser compared to the coiled-coil component, suggests a higher level of stability in Streptavidin. This stability is crucial for its practical application, especially in the context of its utilization as a vital component in test paper detection systems.
Conclusion
The Streptavidin component demonstrated superior stability under the simulated conditions of a 25℃ aqueous solution. Additionally, Streptavidin exhibits a strong affinity for biotin, allowing for effective biotinylation of various antibodies to detect a diverse range of antigens. This feature facilitates convenient modular replacement. As a result, we opted to select Streptavidin as a key component in our detection system and proceeded to construct plasmids for further experimental investigations.
