[gmx-users] Protein in GdmCl solution

Biswajit Gorai biswajit.ju at gmail.com
Mon Feb 18 15:54:07 CET 2013


Dear Justin,

Thanks a lot for your reply.

As u mentioned, I edited the mass as follows:

[ atoms ]
;   nr       type  resnr residue  atom   cgnr     charge       mass
typeB    ch
argeB
      1         CA      1    GDM     C1      1    0.99610  12.010000
      2         N2      1    GDM     N1      2   -0.94930  14.010000
      3         H       1    GDM     H1      3    0.47530   1.008000
      4         H       1    GDM     H2      4    0.47530   1.008000
      5         N2      1    GDM     N2      5   -0.94930  14.010000
      6         H      1    GDM     H3      6    0.47530   1.008000
      7         H      1    GDM     H4      7    0.47530   1.008000
      8         N2      1    GDM     N3      8   -0.94930  14.010000
      9         H      1    GDM     H5      9    0.47530   1.008000
     10         H      1    GDM     H6     10    0.47530   1.008000


I am dealing with a small basic protein of 60 aa.
It is a very stable protein which maintains its structure even at 8 M Urea
but 5M GdmCl can denature it (experimentally).
In-silico (simulation) studies have done in hundreds of ns to denature
their target proteins using chemical denaturants.

Unfortunately, I don't have such computational power.
I already denatured my protein at 423 K around ~40 ns.
So combination of both (chemical and physical denaturants) should able to
produce the expected result
atleast below 40 ns or so (within available resources).
But that is not happening, most surprisingly, even the temperature effect
nullifies.
Showing GdmCl producing counteraction to temperature, which experimentally
doesn't valid for my target.

Looking forward for your reply.

Thanking you.
Biswajit



On Mon, Feb 18, 2013 at 6:55 PM, Justin Lemkul <jalemkul at vt.edu> wrote:

>
>
> On 2/18/13 6:29 AM, Biswajit Gorai wrote:
>
>> Dear GMX Users,
>>
>> Past few months I am struggling to unfold my protein using Guanidinium
>> (GDM) solutions (3,4,5,6 M).
>> I already did temperature (upto 498 K) induced unfolding, and able to get
>> expected results in 60 ns.
>> For GDM (6M), I did simulation upto 120 ns but my target protein is intact
>> (rmsd ~2 A).
>> It seems chemical denaturants take comparatively more time, so I increased
>> the temperature to 423 K to speed-up the process.
>> Now m worried, even after 80 ns simulation in 6M GDM and 423 K, target not
>> showing any remarkable structural change.
>> Also I tried by changing the temperature coupling groups, such as:
>> a) Protein    Non-Protein
>>
>
> I would stick with (a) here, as there is no definitive reason to change it.
>
>  b) System
>> c) Protein+GDM     Water+Ions
>>
>> But all seems waste.
>>
>> *Brief workflow of my work is:*
>>
>> a) Build GDM topology from AmberTools and partial charge was imported
>> from *J.
>>
>> Phys. Chem. B 2011, 115, 12521–1252.
>>
>> gdm.itp::
>>
>> [ moleculetype ]
>> ; Name            nrexcl
>> GDM             3
>>
>> [ atoms ]
>> ;   nr       type  resnr residue  atom   cgnr     charge       mass
>> typeB    ch
>> argeB
>>       1         CA      1    GDM     C1      1    0.99610  12.000000
>>       2         N2      1    GDM     N1      2   -0.94930  14.000000
>>       3         H       1    GDM     H1      3    0.47530   1.000000
>>       4         H       1    GDM     H2      4    0.47530   1.000000
>>       5         N2      1    GDM     N2      5   -0.94930  14.000000
>>       6         H      1    GDM     H3      6    0.47530   1.000000
>>       7         H      1    GDM     H4      7    0.47530   1.000000
>>       8         N2      1    GDM     N3      8   -0.94930  14.000000
>>       9         H      1    GDM     H5      9    0.47530   1.000000
>>      10         H      1    GDM     H6     10    0.47530   1.000000
>>
>>
> The masses on all your atoms are incorrect here.  Check atomtypes.atp for
> your force field for correct values.
>
>  [ bonds ]
>> ;  ai    aj funct  r  k
>>      2     3     1  1.0140e-01  3.3572e+05
>>      2     4     1  1.0140e-01  3.3572e+05
>>      5     6     1  1.0140e-01  3.3572e+05
>>      5     7     1  1.0140e-01  3.3572e+05
>>      8     9     1  1.0140e-01  3.3572e+05
>>      8    10     1  1.0140e-01  3.3572e+05
>>      1     2     1  1.3390e-01  4.0819e+05
>>      1     5     1  1.3390e-01  4.0819e+05
>>      1     8     1  1.3390e-01  4.0819e+05
>>
>> [ pairs ]
>> ;  ai    aj funct
>>       2      6      1
>>       2      7      1
>>       2      9      1
>>       2     10      1
>>       5      3      1
>>       8      3      1
>>       5      4      1
>>       8      4      1
>>       5      9      1
>>       5     10      1
>>       8      6      1
>>       8      7      1
>>
>> [ angles ]
>> ;  ai    aj    ak funct  theta   cth
>>      1     2     3     1  1.2124e+02  4.0827e+02
>>      1     2     4     1  1.2124e+02  4.0827e+02
>>      1     5     6     1  1.2124e+02  4.0827e+02
>>      1     5     7     1  1.2124e+02  4.0827e+02
>>      1     8     9     1  1.2124e+02  4.0827e+02
>>      1     8    10     1  1.2124e+02  4.0827e+02
>>      3     2     4     1  1.1485e+02  3.3514e+02
>>      6     5     7     1  1.1485e+02  3.3514e+02
>>      9     8    10     1  1.1485e+02  3.3514e+02
>>      2     1     5     1  1.2017e+02  6.1061e+02
>>      2     1     8     1  1.2017e+02  6.1061e+02
>>      5     1     8     1  1.2017e+02  6.1061e+02
>>
>> [ dihedrals ]
>> ;i  j   k  l     func   C0  ...  C5
>>      2    1    5    6      3     5.64840     0.00000    -5.64840
>> 0.00000
>> 0.00000     0.00000     ;
>>      2    1    5    7      3     5.64840     0.00000    -5.64840
>> 0.00000
>> 0.00000     0.00000     ;
>>      2    1    8    9      3     5.64840     0.00000    -5.64840
>> 0.00000
>> 0.00000     0.00000     ;
>>      2    1    8    10     3     5.64840     0.00000    -5.64840
>> 0.00000
>> 0.00000     0.00000     ;
>>      5    1    2    3      3     5.64840     0.00000    -5.64840
>> 0.00000
>> 0.00000     0.00000     ;
>>      8    1    2    3      3     5.64840     0.00000    -5.64840
>> 0.00000
>> 0.00000     0.00000     ;
>>      5    1    2    4      3     5.64840     0.00000    -5.64840
>> 0.00000
>> 0.00000     0.00000     ;
>>      8    1    2    4      3     5.64840     0.00000    -5.64840
>> 0.00000
>> 0.00000     0.00000     ;
>>      5    1    8    9      3     5.64840     0.00000    -5.64840
>> 0.00000
>> 0.00000     0.00000     ;
>>      5    1    8    10     3     5.64840     0.00000    -5.64840
>> 0.00000
>> 0.00000     0.00000     ;
>>      8    1    5    6      3     5.64840     0.00000    -5.64840
>> 0.00000
>> 0.00000     0.00000     ;
>>      8    1    5    7      3     5.64840     0.00000    -5.64840
>> 0.00000
>> 0.00000     0.00000     ;
>>      1    3    2    4      3     9.20480     0.00000    -9.20480
>> 0.00000
>> 0.00000     0.00000     ;
>>      1    6    5    7      3     9.20480     0.00000    -9.20480
>> 0.00000
>> 0.00000     0.00000     ;
>>      1    9    8    10     3     9.20480     0.00000    -9.20480
>> 0.00000
>> 0.00000     0.00000     ;
>>      8    1    5    2      3     9.20480     0.00000    -9.20480
>> 0.00000
>> 0.00000     0.00000     ;
>>
>>
>> *b) Build small equilibrated (NVT at 310 K) boxes at 3, 4, 5, 6 M of GDM+
>>
>> ion.
>> c) Protein in GDM
>>     i) Solvate the protein in 6M GDM
>>     ii) Add CL to neutralize the system
>>     iii) Minimize using SD followed by CG.
>>     iv) NVT (2 ns) and NPT (5 ns) equilibration
>>     v) Finally production run.
>>
>> *md.mdp::*
>>
>> *title           = Gdm-Amber-CTX-6M
>>
>> ; Run parameters
>> integrator      = sd
>> nsteps          = 20000000
>> dt              = 0.002
>> ; Output control
>> nstxout         = 5000
>> nstvout         = 5000
>> nstxtcout       = 5000
>> nstenergy       = 5000
>> nstlog          = 5000
>> ; Bond parameters
>> continuation    = yes
>> constraint_algorithm = lincs
>> constraints     = all-bonds
>> lincs_iter      = 1
>> lincs_order     = 4
>> ; Neighborsearching
>> ns_type         = grid
>> nstlist         = 5
>> rlist           = 1.0
>> rcoulomb        = 1.0
>> rvdw            = 1.0
>> ; Electrostatics
>> coulombtype     = PME
>> pme_order       = 4
>> fourierspacing  = 0.16
>> ; Temperature coupling is on
>> tcoupl          = V-rescale
>> tc-grps         = protein_gdm Water_and_ions    ; Protein Non-Protein  ;
>> System (also tried)
>> urate
>> tau_t           = 0.1   0.1
>> ref_t           = 423   423
>> ; Pressure coupling is on
>> pcoupl          = Parrinello-Rahman
>> pcoupltype      = isotropic
>> tau_p           = 5.0
>> ref_p           = 1.0
>> compressibility = 4.5e-5
>> ; Periodic boundary conditions
>> pbc             = xyz
>> ; Dispersion correction
>> DispCorr        = EnerPres
>> ; Velocity generation
>> gen_vel         = no *
>>
>>
>>
>> I am using AMBER99SB-ILDN and TIP3P in GROMACS 4.5.4v. I really need the
>> valuable sugestions.
>>
>
> What does your assessment of the literature tell you?  Denaturation
> simulations have been done before with a variety of chemicals like urea and
> SDS.  How long did they take and what were the simulation conditions?  Is
> your protein comparable in size to others that have been assessed before,
> or is it much larger (thus implying it would take longer)?  What makes you
> think that chemical denaturation would be observed on the time scale of
> tens of ns, when in reality it may take far longer?
>
> -Justin
>
> --
> ==============================**==========
>
> Justin A. Lemkul, Ph.D.
> Research Scientist
> Department of Biochemistry
> Virginia Tech
> Blacksburg, VA
> jalemkul[at]vt.edu | (540) 231-9080
> http://www.bevanlab.biochem.**vt.edu/Pages/Personal/justin<http://www.bevanlab.biochem.vt.edu/Pages/Personal/justin>
>
> ==============================**==========
> --
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