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1. Backbone Deviation and Structural Shifts

• The N501Y mutation induces a significant backbone shift compared to the wild-type (N501) and other mutations, such as N501S and N501T. The root-mean-square deviation (RMSD) values for N501Y (0.047 nm) and N501T (0.04 nm) were higher than those for the wild-type, indicating a greater structural change in the N501Y variant.
• These shifts were not uniform over time, with peak deviations observed at different simulation times: N501Y reached its peak deviation of 0.49 nm at 6.78 ns, while other variants peaked at different times and with smaller deviations.
• Despite the variability in deviations, all variants eventually reached an equilibrium state after 42.3 ns, with backbone oscillations staying below 0.15 nm during the last phase of the simulation (42.3-50 ns), confirming system stability.

2. Solubility and Surface Accessibility

• The solvent-accessible surface area (SASA) analysis showed that N501Y and N501S had similar solubility profiles, with N501S exhibiting a slightly higher SASA (364.66 ± 3.28 nm²) compared to the others, suggesting it might contribute more favorably to protein solubility.
• Interestingly, while N501T did not significantly alter the surface accessibility, N501Y showed slight changes in solubility, particularly around the 37.5 ns mark, where N501S had a higher SASA score than N501Y.

3. RMSF Analysis and Residue Fluctuations

• The root mean square fluctuation (RMSF) analysis, which measures residue flexibility, showed that the N501Y variant exhibited higher fluctuation in the RBD domain [499-505], particularly around Pro-Thr-Tyr-Gly-Val-Gly-Tyr. This behavior was unique to the N501Y mutant, indicating that Tyrosine at position 501 induced local instability in the RBD domain.
• Other variants, N501S and N501T, showed similar trends with lower RMSF values in specific regions, such as ValLeuTyrAsnSerAlaSer in the S-protein RBD [367-373], indicating less flexibility compared to N501Y.

4. Secondary Structure Analysis

• The secondary structure prediction revealed distinct conformational changes between N501 and N501Y. For example:
  • In N501, the α-helix formed in the RBD domain [366-370] was stable, but in N501Y, there were frequent transitions from α-helix to π-helix, particularly after 38.5 ns.
  • Additionally, a 3/10-helix structure in N501 at the RBD domain [503-505] was destabilized in N501Y, which shifted to a turn at the same interval.
• These structural transitions suggest that N501Y may destabilize some stable conformations in the RBD domain, potentially affecting the spike protein's ability to bind to ACE2 effectively.

5. Hydrogen Bond Dynamics

• The analysis of hydrogen bonds between the spike protein RBD and ACE2 highlighted notable differences in polar contacts for the N501Y mutant:
  • The N501Y mutation enhanced the strength and persistence of hydrogen bonds compared to the wild-type structure, particularly in the RBD domain [499-505].
  • A specific hydrogen bond between Gly502 (spike protein) and Lys353 (ACE2) was more stable in N501Y than in N501, with the N501Y variant showing a 98.4% occupancy during the convergence phase, compared to 79.5% for N501.
  • The Tyr505 (spike protein) to Glu37 (ACE2) hydrogen bond was also more persistent in N501Y, with a stronger interaction (occupancy increased by 10.6% compared to N501).
  • Thr500 in the spike protein also formed different hydrogen bonds in N501Y: while it usually interacted with Asp355 (ACE2) in the wild type, it increasingly formed a bond with Tyr41 (ACE2) in N501Y, compensating for the reduced interaction with Asp355.

6. Binding Energy and Affinity

• Binding energy shifts due to the N501Y mutation were calculated for the most affected hydrogen bonds. The mutation caused an increase in binding affinity, with a shift of 0.91 kcal/mol during the simulation and 1.06 kcal/mol during the convergence phase. This indicates a stronger and more stable interaction between the spike protein RBD and ACE2 in the N501Y variant.

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