Methodological Decision Log
Project: MACS
Document type: Technical Documentation
Year: 2026
Research status: Computational Discovery
Motif finalized as HALDSLE
Decision: MACS will use HALDSLE repeats instead of HALKSLE.
Reason: The intended pH-switch mechanism depends on Asp/Glu-assisted histidine pKa engineering. Aspartate provides a local negative electrostatic environment that may increase histidine protonation propensity near the target pH threshold around 6.8. Lysine would introduce a positive charge and may oppose histidine protonation, weakening the intended histidine-zipper mechanism.
Alternative considered: HALKSLE, which appeared in earlier sequence files and AlphaFold input.
Risk / limitation: The pKa shift is a design hypothesis. HALDSLE must be validated by PROPKA/H++ and MD. It must not be assumed correct solely from design intuition.
pH 6.8 histidine protonation candidate set
Decision: For pH 6.8, do not protonate all histidines. Use a selective borderline histidine set based on PROPKA pKa values and structural inspection.
Primary pH 6.8 candidates: A:282, A:289, A:296, B:272, B:293, B:324, B:329, B:332.
Reason: These residues have PROPKA-predicted pKa values near the target pH range and/or are structurally close to the HALDSLE/interface region.
pH 7.4 rule: Histidines will remain neutral unless a strong residue-specific reason exists.
Risk / limitation: Static PROPKA on an AlphaFold model cannot capture dynamic pKa shifts. This is a fixed-protonation approximation, not constant-pH MD.
pH 7.4 histidine assignment
Decision: All histidines in the pH 7.4 model were assigned as neutral HIE.
Reason: pH 7.4 is above the predicted pKa values for most histidines. Therefore, doubly protonated HIP was not used.
Alternative considered: Mixed HID/HIE tautomer assignment. Deferred for simplicity; this model uses a uniform neutral HIE assignment.
Risk / limitation: HIE/HID tautomer choice can affect local hydrogen-bonding. This should be acknowledged as a fixed-protonation approximation.
pH 6.8 topology finalized
Decision: pH 6.8 topology was generated with selective HIP assignments: A:282, A:289, A:296, B:272, B:293, B:324, B:329, B:332. All other histidines were assigned as neutral HIE.
Reason: Selected residues are borderline pKa/interface/motif candidates based on PROPKA and structural inspection.
Risk / limitation: This is still a fixed-protonation model. It approximates pH-dependent behavior but does not replace constant-pH MD.
Fixed-protonation approximation for pH 6.8
Decision: The pH 6.8 model uses selective HIP assignments for eight borderline histidines.
Reason: PROPKA/Henderson–Hasselbalch analysis indicates that these residues have higher protonation propensity at pH 6.8 than at pH 7.4. The average predicted protonated fraction for the selected set is substantially higher at pH 6.8 than at pH 7.4, supporting a pH-dependent protonation gradient.
Alternative considered: Constant-pH MD, which would allow dynamic protonation/deprotonation. This is more rigorous but outside the current compute/software scope.
Important limitation: Classical fixed-protonation GROMACS MD cannot represent partial protonation. Assigning these residues as HIP does not mean that they are experimentally known to be 100% protonated at pH 6.8. Rather, this is a discrete approximation used to test whether protonation of the most pH-sensitive HALDSLE-associated histidines can drive the intended conformational switch. In any report or manuscript, this model must be described as a fixed-protonation approximation, not as direct proof of physiological full protonation at pH 6.8.
Local 1 ns validation completed
Decision: Both pH 6.8 and pH 7.4 systems passed local 1 ns validation MD after EM/NVT/NPT.
Results: Both trajectories completed successfully, produced final coordinates, and passed standard validation checks.
Interpretation: The systems are stable enough to proceed to longer production MD.
Risk / limitation: 1 ns validation only detects gross setup instability. It does not prove long-timescale convergence or biological correctness.
GROMACS 2026.1 production TPR regeneration
Decision: Final 300 ns production TPR files were regenerated with GROMACS 2026.1.
Result: Both pH 6.8 and pH 7.4 300 ns TPR files were generated successfully with GROMACS 2026.1.
Output policy: TRR output remains disabled. Compressed XTC output is used for trajectory storage.