Molecular Dynamics Simulations Analysis Hemochromatosis

Hemochromatosis>>Task 10: Molecular dynamics simulations analysis

Short task description

Detailed description: Molecular dynamics simulations analysis

Protocol

A protocol with a description of the data acquisition and other scripts used for this task is available here.

Dummy

Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is depicted under LINKTOMINDISTTODO.

Calculation statistics

<figtable id="tab:simulation_stats"> Statistics of the MD simulations

Input	Calc. time	Calc. speed	time to reach 1 s
Wildtype	13h31:15	17.750 ns/day	154350,8 years
C282S	13h35:05	17.667 ns/day	155075,9 years
R224W	13h35:02	17.668 ns/day	155067,1 years

</figtable>

GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.

Energies

Pressure

**Table 2:** different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)

</figtable>

The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).

Temperature

**Table 3:** different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.

Potential

**Table 4:** different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:

The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).

Total energy

**Table 5:** different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.

All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.

Minimum distance between periodic boundary cells

**Table 6:** different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.

**Table 7:** different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

The resulting minimal distances can be seen in TABLETODO. We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.

RMSF for protein and C-alpha

Protein based

**Table 8:** different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

In general the RMS fluctuations of all three models look similar. The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peat at around residue 20.

C-Alpha based

**Table 9:** different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

The c-alpha based RMSF shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.

Statistical values

With the GIVENSCRIPTTODO we calculated the values for the t-Test:

1a6zC to R224W : 8.079405e-57**

1a6zC to C282S : 2.87453e-56**

R224W to C282S : 5.45069e-25**

Pymol analysis of average and bfactor

**Table 11:** Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.

As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).

Radius of gyration

**Table 12:** different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

**Table 13:** different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

These graphs show the radii of gyration in general als well as in each dimension. From start to end there is a slight increase in general gyrationradius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.

Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.

solvent accessible surface area

**Table 14:** display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

**Table 15:** display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

hydrogen-bonds between protein and protein / protein and water

Protein-Protein

**Table 16:** the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.

Protein-Water

**Table 17:** the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.

In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.

The R224W model shows a different behavior over time:

The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm.

The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:

For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.

Ramachandran plots

**Table 18:** Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are pretty filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.

RMSD matrix

**Table 19:** RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

**Table 20:** RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)

</figtable>

The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.

The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.

The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.

The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.