Thermostable alpha-amylase molecular dynamics

Undergraduate chemistry thesis analyzing the thermostability mechanism of Aspergillus oryzae alpha-amylase mutants E156D, D157N, and D448S using molecular dynamics simulation.

Protein Modeling
Molecular Dynamics
Simulation Data Analysis
Research Writing

Thesis focus

Explaining why the mutant enzyme stays more stable at heat

This thesis studied Aspergillus oryzae alpha-amylase, commonly referred to as TAKA, because thermostable alpha-amylase is valuable in starch processing, food and beverage, pharmaceutical, textile, animal feed, and detergent applications.

The research compared native TAKA with a mutant containing E156D, D157N, and D448S. E156D and D157N sit around domain B, while D448S sits around domain C. The main question was whether these substitutions create local interactions that make the protein more rigid under high temperature simulation.

Extracted molecular structure map showing E156D, D157N, and D448S mutation positions — Mutation positions extracted from the thesis structure figure.

Mutation positions extracted from the thesis structure figure.

Simulation setup

Native TAKA was prepared from the 6TAA structure, then the mutant model was built by introducing E156D, D157N, and D448S. Systems were protonated at pH 6.0, solvated with TIP3P water, parameterized with FF14SB, minimized, heated, equilibrated, and run for 100 ns.

Analysis workflow

The MD output was interpreted using RMSD, SASA, radius of gyration, RMSF, trajectory alignment, and hydrogen-bond interaction analysis. Supporting tools included AMBER, AmberTools23, BIOVIA Discovery Studio, UCSF Chimera, and VMD.

Result 01

RMSD showed the mutant resisted global structural drift

RMSD was used to monitor how far the backbone moved from the reference structure during simulation. At 300 K, native and mutant TAKA stayed close, which made it a useful control condition.

At 350 K and especially 400 K, native TAKA became more unstable. The native trajectory rose more sharply late in the run, while the mutant curve stayed lower and less extreme. This supported the interpretation that the mutations reduced large conformational deviation at heat.

Extracted RMSD graph at 300 K for native and mutant TAKA — 300 K

Extracted RMSD graph at 350 K for native and mutant TAKA — 350 K

Extracted RMSD graph at 400 K for native and mutant TAKA — 400 K

Result 02

SASA indicated lower solvent exposure in the mutant

SASA was used to observe how much protein surface became accessible to solvent. Higher and more fluctuating SASA can indicate that the structure is opening or exposing more surface area.

The native enzyme showed stronger fluctuation, including noticeable exposure increases at elevated temperature. The mutant trend was more controlled, suggesting that the protein surface stayed less disrupted during heating.

Extracted SASA graph at 300 K for native and mutant TAKA — 300 K

Extracted SASA graph at 350 K for native and mutant TAKA — 350 K

Extracted SASA graph at 400 K for native and mutant TAKA — 400 K

Result 03

Radius of gyration supported better compactness

Radius of gyration measures compactness. A lower and steadier Rg value suggests that the folded protein remains more compact instead of expanding toward an unfolded state.

At 350 K and 400 K, native TAKA showed larger late-simulation increases, while mutant TAKA remained comparatively stable. This matched the RMSD and SASA interpretation: the mutant behaved as a more compact high-temperature structure.

Extracted radius of gyration graph at 300 K for native and mutant TAKA — 300 K

Extracted radius of gyration graph at 350 K for native and mutant TAKA — 350 K

Extracted radius of gyration graph at 400 K for native and mutant TAKA — 400 K

Result 04

RMSF localized the stability difference to flexible loops

RMSF was used to read flexibility residue by residue. Loop regions are especially important because high loop mobility can initiate local unfolding.

The thesis focused on loops around residues 150-160 in domain B and 448-455 in domain C. These regions are near the mutation sites and became the structural explanation for why global metrics favored the mutant.

Extracted RMSF graph at 300 K for native and mutant TAKA — 300 K

Extracted RMSF graph at 350 K for native and mutant TAKA — 350 K

Extracted RMSF graph at 400 K for native and mutant TAKA — 400 K

Result 05

Trajectory alignment showed calmer motion around domains B and C

Structures sampled every 20 ns were aligned to compare how native and mutant proteins moved through time. The extracted figures show the mutant loops staying more gathered, while corresponding native regions appear more spread across the trajectory.

This visual result connected the numerical metrics to an actual structural mechanism: the mutations did not merely change a graph; they changed local loop behavior in the regions connected to thermostability.

Extracted trajectory alignment of mutant TAKA around domain B — Domain B mutant

Extracted trajectory alignment of native TAKA around domain B — Domain B native

Extracted trajectory alignment of native TAKA around domain C — Domain C native

Extracted trajectory alignment of mutant TAKA around domain C — Domain C mutant

Result 06

E156D and D157N added local hydrogen-bond support in domain B

In native TAKA, the domain B loop had fewer stabilizing contacts around residues 156 and 157. After mutation, D156 and N157 formed a richer local interaction pattern with surrounding residues such as Q160, N157, N154, and T159.

These added hydrogen-bond interactions help explain why the domain B loop became less flexible. In practical terms, the substitutions made a local section of the enzyme harder to loosen during high-temperature simulation.

Extracted hydrogen bond visualization for native TAKA around domain B — Native

Extracted hydrogen bond visualization for mutant TAKA around E156D and D157N — Mutant E156D and D157N

Result 07

D448S strengthened the domain C loop through new interaction

D448S was analyzed around the domain C loop. The mutant residue S448 formed hydrogen-bond interaction with nearby N450, while the native D448 region did not show the same stabilizing pattern.

This added interaction supported the RMSF interpretation at residues 448-455: the mutant loop became more locally stable, contributing to the overall rigidity observed in the high-temperature MD results.

Extracted hydrogen bond visualization for native TAKA around D448 — Native D448

Extracted hydrogen bond visualization for mutant TAKA around D448S — Mutant D448S

Conclusion

The main mechanism proposed by the thesis is that E156D, D157N, and D448S improve thermostability by adding stabilizing hydrogen-bond interactions around domains B and C. These local changes reduce loop flexibility, preserve compactness, lower excessive solvent exposure, and make the mutant structure more rigid at high temperature.

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