Chimeric Protein Construction
The retrieved amino acid sequences of ScFv and IFN-β were fused together by a linker of 15 amino acid to form ScFv-IFN-β construct. The designed ScFv-INF-β construct contains ScFv structure comprising 242 amino acids residues with a linker of 14 amino acids that connect both variable regions, VL and VH. One methionine, one glycine and two serine were also incorporated at the N-terminal of the ScFv to reduce the chances of frameshift mutation, followed by a polyHistidine tag (His6). The IFN-β block of the construct contain 167 amino acids residues. Considering the complete sequence of the ScFv- IFN-β, the final construct would be 444 amino acids residues in length as shown in Figure 1.
Physicochemical Properties and Solubility
The physiochemical characteristics and solubility feature of ScFv-IFN-β were analyzed by ProtParam and SOLpro (Scratch Protein Detector) online servers respectively. The results predicted were related to various properties of the protein. The molecular weight of the design construct was computed as 48172.96 Da. The GRAVY and aliphatic index value was calculated as -3.777 and 73.38 respectively. While the theoretical PI and net charge were 9.02, and +5 respectively. Based on the solubility analysis, ScFv-IFN-β was predicted as a soluble protein with a value of 0.634.
3D Model Building, Refinement, and Validation
The 3D structures of ScFv, IFN-β, ScFv-IFN-β fusion protein and HER1 receptor were constructed by AlphaFold server as shown in Figure 2(A-D). A model having the highest confidence-score (c-score) was chosen from 5 predicted models.
Confidence score of the selected model was between the 5 and 2 (the higher the confidence score, higher confidence and vice versa). For refinement, the predicted 3D model was submitted to Galaxy Refine server and 5 refined models were proposed. Furthermore, among these 5 refined models, one refined model with the highest favored regions in the Ramachandran plot was adopted as shown in Figure 3. The quality of the refined and unrefined models were analyzed by PROCHECK and RAMPAGE.
Allergenicity Evaluation
AlgPred web server was employed to determine the allergencity of the ScFv-IFN-β. According to AlgPred results, our construct was predicted as non-allergenic (Score=-0.74675496 with Threshold=-0.40) for human body upon consumption.
mRNA Stability
To predict mRNA stability, RNA fold web server was employed that measures the free minimization energy. The free energy was calculated as -534.50 kcal/mol as shown in Figure 4. The negative value of free energy indicates stable structure of RNA molecule.
Protein-Protein Docking
To evaluate the binding affinity of the protein molecules, we performed docking of 6 protein complexes with each other that were categorized into 6 groups. Some particular parameters like Docking score, Confidence score, and Ligand RMSD (Å) were considered as significance values in the results as shown in Table 2.
Table 2: Molecular docking of protein complexes
S No.
|
Docked complexes
|
Docking score
|
Confidence score
|
Ligand RMSD (Å)
|
1
|
HER1 receptor vs ScFv
|
-259
|
0.89
|
45.89
|
2
|
HER1 receptor vs ScFv-IFN-β
|
-285.3
|
0.94
|
53.23
|
3
|
ScFv-IFN-β vs IFNR1
|
-255.9
|
0.89
|
86.07
|
4
|
ScFv-IFN-β vs IFNR2
|
-255.5
|
0.89
|
64.31
|
5
|
IFN-β vs IFNR1
|
-390.7
|
0.99
|
1.03
|
6
|
IFN-β vs IFNR2
|
-456
|
0.99
|
0.6
|
Abbreviations. HER1; Hormonal Epidermal Receptor 1, ScFv; Small Chain Fragment Variable, INF-β; Interferon beta, IFNR; Interferon Receptor
Overall, our final construct, ScFv-IFN-β fusion protein, shows high binding affinity and specificity for HER1 receptor) as shown in Figure 5 (A & B). All other visualized models of protein complexes are given in Figures (S1-S5) in supplementary material.
Molecular dynamics simulation
The resultant model shows better deformation at each amino acids capacity level. The Eigon value is 1.747149e-06, indicating less stiffness and more deformation. Highly correlated regions in heat maps and low RMSD value shows better interactions of the individual amino acids (Figure 6). The figure (6) depicts the explanatory results obtained from IModS molecular dynamics simulation analysis. In Fig. 6; (A) the B-factor is indicated, (B) part indicates deformability which shows low level of deformation at all the amino acid residues, (C) Eigon values are indicated as 1.747149e-06, and (D) indicates the variance shown in both red and green color, (E) and (F) indicate co-variance and elastic network of the complex as well.