Journal of Nano Chemical Agriculture , Vol(1) , No(3)
80
PH Effects On 273th Codon Of P53 Gene By Computational Methods
Nastaran Asghari Moghaddam1
Department of Biology, College of Basic Science, Tehran Science and Research Branch, Islamic Azad
University, Tehran, Iran
namoghadam@gmail.com
Abstract
P53 tumor suppressor gene, also known as “genome guardian” is mutated in more than half of all kind of
cancers. In this study we have investigated the controls of environmental pH for P53 gene mutation in point of
specific sequence which is prone to mutagenesis. The most probable cancerous mutations occur as point
mutations in exons 5 -8 of P53 gene. By experimental research, it is revealed that acidic pH raised the rate of
cancer and mutation in 273CGT codon of P53 gene. It to some extent is due to protonation of this three
nucleotide codon. Mutation in this codon changes the encoding amino acid and subsequently produces a
protein which has oncogenic features instead of tumor suppressor characteristics of original p53 protein. In
current study, we perform investigation on the impact of protonation on stability of codon 273CGT in this gene.
We used HYPERCHEM software for answering to our mentioned goal above.Our results suggested a reliable
answer about the effect of protonation on mentioned codon and its stability. From theoretical point of view,
acidity can increase the unstability of this specific codon. Along with the experimental investigations, our
results can to some extent elaborate the mutagenesis of acidic pH.
Keywords: P53 gene, protonation, mutagenesis
Introduction
P53 is a tumor-suppressor protein which has
classical features of transcription factor. It
responses to different cellular stress via inducing
activation or repression of more than 2500 genes
[2]. Because of its critical role in protection against
cancer, it is called “the guardian of genome”.
Somatic mutation of this gene has been observed
in more than 50 percent of different human
cancers [16]. Unlike other tumor suppressor genes
(like RB, APC and BRCA1) in which inactivation
occurs by frameshift and nonsense mutations,
about 80 percent of P53 mutations are missense
[7]. In P53 gene the most prevalent site of
mutagenesis is along 5-8 exons, which encodes
DNA binding domain. For instance, codon 273
(with 3 nucleotide sequence of CGT) included 8.7
percent of all mutations in P53 gene [9].
The importance of studying mutational pattern is
to better understand the function of p53 domains,
their effects on tumor- suppressing properties of
p53 and the nature of etiologic substances as
environmental etiologic biomarkers [13].
In addition, observations suggest that the pH of
tumor microenvironment is more acidic than
normal cells [4]. It is shown that disease “Barrett’s
esophagus” is related to acid reflux [15] and even
in its early stage mutation in P53 has been
observed [5].
81
PH Effects On 273th Codon Of P53 Gene
Although the role of acidic pH is significant in
carcinogenesis, its molecular mechanism is little
known. It is known that physical properties of DNA
is crucial for molecular genetic studies [10]. DNA
molecule is constantly exposed to a great range of
physical and chemical substances which harm its
structure [14].
The effect of pH on DNA structure is not fully
evaluated, because of the difficulty of measuring
this quantity in cellular nucleus. In this regard,
simulation and computational chemistry could be
helpful. In current study, we tried to evaluate a
new carcinogenesis pathway caused by pH
alteration and creates missense mutation. We
specifically focused on 273 codon (figure 1) of P53
gene.
Materials and Methods
CGT three-nucleotide was drawn as a double
stranded DNA structure by HyperChem™. Then the
structure was optimized by geometric optimization
order. To determine the effect of pH on this
structure a periodic box with 30 °A dimensions was
designed. In addition, according to pH
corresponding H+ and OH- for pH values 6.8 and 7.4
were respectively located within a periodic box.
Simulation was done in MM+, AMBER, BIO+ and
OPLS force fields.
Molecular Mechanics
calculations were assessed by Monte Carlo method
[12]. Three important energy parameters – kinetic
energy, potential energy and total energy- in four
different simulating temperatures (308, 310, 312
and 314 Kelvin) were used for computation.
Results and Discussion
More than half of all human cancers have
mutation in P53 gene. Theoretically evaluation of
environmental factors like pH can help us to
understand the causes of mutation and its
molecular mechanisms. In current study
computations were done in sophisticated and
appropriate molecular modeling environment of
HyperChem™ which is well known for its quality and
flexibility [6, 11]. It is known that atoms are held
together by forces. Function of biological systems
arises from interaction of resilient bonds between
atoms and electron motion. The main purpose is to
seek for the lowest energy, in which the molecule
is in its most stable state [8, 17]. In this study
AMBER, MM+, BIO+ and OPLS force fields were
chosen. When CGT is modeled, it undergoes
shaking, rotating, stretching, and etc. functions
around its bonds. The total potential energy is the
sum of mentioned contribution interactions based
on the force fields.
Therefore, force fields are a series of functional
energy parameters that evaluate performance and
calculate the potential energy of molecule in
various positions of its constituent atoms and
bonds [3].
MM+ is a proper parameter for attaining vibration
motion of atoms, related bond stretching
potential, and angles bending. AMBER force field
has extensive application for proteins and nucleic
acids. It assigns all conformational energies and
treats with hydrogen bond energy, and torsion
term [1]. Like AMBER, OPLS is designed for
computation of proteins and nucleic acids. In this
force field bonded potentials are similar to AMBER
and its non-bonded potentials involves vander
waals and electrostatics. BIO+ filed is an extended
form of CHARMM. Similar to AMBER and OPLS it
has been designed to study macromolecules [8].
CGT codon was simulated in mentioned force fields
in 4 different temperature (308K, 310K, 312K and
314K). To elucidate the effect of CGT energy on
molecular mechanic calculation, the most usual
expression for total potential energy is given by the
following equation:
82
Journal of Nano Chemical Agriculture , Vol(1) , No(3)
𝑏𝑜𝑛𝑑𝑠
𝐸
𝑡𝑜𝑡𝑎𝑙
= ∑ 𝐸𝑖𝑠𝑡𝑟𝑒𝑡𝑐ℎ
𝑖
𝑏𝑜𝑛𝑑𝑎𝑛𝑔𝑙𝑒𝑠
+
∑
𝐸𝑖𝑏𝑒𝑛𝑑
𝑖
𝑑𝑖ℎ𝑒𝑑𝑟𝑎𝑙𝑎𝑛𝑔𝑙𝑒𝑠
+
∑
𝑎𝑡𝑜𝑚𝑝𝑎𝑖𝑟𝑠
𝐸𝑖𝑡𝑜𝑟𝑠𝑖𝑜𝑛 +
∑
𝑖
𝑖𝑗
Covalent Interactions
𝐸 𝑣𝑎𝑛𝑑𝑒𝑟𝑤𝑎𝑎𝑙𝑠 +
∑
𝐸 𝑒𝑙𝑒𝑐𝑡𝑟𝑜𝑠𝑡𝑎𝑡𝑖𝑐𝑠
𝑖𝑗
Non- Covalent Interactions
E total is the sum of bonded and non-bonded
interactions
Ebond is stretching bond energy between two atoms
Eangle is energy of bending an angle
Etorsion is torsion energy of rotation around a bond
E electrostatic and Evander waals are two energies which
are exponent distribution, and repulsion or
attraction
between
non-bonded
atoms,
respectively.
The other two calculated energy quantities are
kinetic and total energy values. In symbols the
total energy equals:
𝐸𝑡𝑜𝑡𝑎𝑙 = ∑ 𝐸𝑝𝑜𝑡𝑒𝑛𝑡𝑖𝑎𝑙 + ∑ 𝐸𝑘𝑖𝑛𝑒𝑡𝑖𝑐
𝑎𝑡𝑜𝑚𝑝𝑎𝑖𝑟𝑠
(2)
From a statistical point of view, the obtained
valuable data for three basis sets of
thermodynamic parameters (Ekinetic, Epotential, Etotal),
analyzed under the different simulation procedure,
various temperatures and different pH values
every 10 ps span are listed in tables1, 2 and 3.
According to results observed in table 1, the
amount of kinetic energy constantly increase as
the temperature was been raised. Obtained figures
for kinetic energy in different time steps and
various force fields were constant and the
maximum quantity observed in314 K, 177.83 and
(1)
213.4 Kcal/mol for pH values of 7.4 and 6.8,
respectively. In acidic condition the amount of
kinetic energy was approximately 25 percent more
than physiologic pH. It is known that to have
optimum function in biologic system, the energy
levels must be in the minimum level.
By comparing data from different pH values (see
table 2), it is clear that potential energy of acidic
pH is higher than physiologic pH and this increase
can be observed in different force fields. In figure 2
minimum potential energy calculated by AMBER
force filed in pH 7.4 and 6.8 have been reported.
The increase of potential energy can be observed
in this graph. It is notable that molecular stability in
physiologic pH is more. Minimum potential energy
levels in normal body temperature were 1918.23
and 289.57 for pH 6.8 and 7.4, respectively. Data
analysis of table 3 exhibited that total energy
quantities were affected by reducing pH value and
increasing temperature.
Current study was done to evaluate the
thermodynamic role of acidic pH in creation of
mutation in 273th codon of P53 gene. Analysis of
kinetic, potential and total energies in 4 molecular
mechanic energy fields. Increase of energy level
has been observed in all three mentioned
83
PH Effects On 273th Codon Of P53 Gene
parameters. Energy increase leads to molecular
instability. According to the obtained results in this
study, it can be concluded that acidic condition in
cellular environment directly produce unstable
codon structure which can cause changes in codon
structure. If this alteration in biologic system is not
recognized and not repaired, it can result in the
mutation of DNA. Regarding to the fact that
genetic changes’ negative role in cells, this can be a
beginning point for cancer progression. The results
can be a cancer with a poor prognosis if this
alteration happens in a gene like P53.
References
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Table 1. Computed CGT Kinetic energy ( kcal/ mol ), belong to AMBER, MM+, BIO+ and OPLS force fields under
four different temperature and 2 various pH values.
Kinetic Energy (K Cal/mol)
6.8
pH condition
7.4
Method
308 K
310 K
312 K
314 K
308 K
310 K
312 K
314 K
AMBER
209.3228
210.6821
212.0413
213.4005
174.4357
175.5684
176.7011
177.8338
BIO
+
209.3228
210.6821
212.0413
213.4005
174.4357
175.5684
176.7011
177.8338
MM
+
209.3228
210.6821
212.0413
213.4005
174.4357
175.5684
176.7011
177.8338
OPLS
209.3228
210.6821
212.0413
213.4005
174.4357
175.5684
176.7011
177.8338
Table 2. Computed CGT potential energy ( kcal/ mol ), belong to AMBER, MM+, BIO+ and OPLS force fields
under four different temperature and 2 various pH values.
Potential Energy (Kcal/mol)
Amber/Monte Carlo
6.8
Method
pH condition
time
(PS)
308 K
310 K
10
20
30
40
50
60
70
80
90
100
42186.5
10721.1
6389.2
4700.9
3792.5
3159.3
2751.5
2432.0
2154.7
1955.1
308 K
Method
pH condition
time
(PS)
10
20
30
40
50
60
70
80
90
100
310 K
312 K
MM+/ Monte Carlo
7.4
314 K
308 K
310 K
312 K
314 K
308 K
39561.2 30530.5
9311.3 8789.2
5942.6 5670.5
4604.6 4428.1
3764.8 3643.5
3172.1 3035.1
2761.0 2625.5
2447.3 2307.0
2198.2 2098.2
2002.8 1928.2
BIO/Monte Carlo
6.8
33473.4
9425.3
6103.5
4699.8
3861.3
3318.5
2875.8
2533.6
2246.3
2055.2
781.4
515.8
416.0
389.3
359.7
339.7
327.6
309.2
297.0
303.0
805.8
783.7
522.4
522.9
398.9
447.6
354.7
396.9
337.0
371.4
338.5
340.6
326.8
322.6
321.2
311.9
319.7
310.0
303.2
292.9
BIO/Monte Carlo
7.4
822.3
559.7
448.1
396.5
371.0
342.9
334.6
332.4
322.5
325.8
11706.2
7401.4
5039.8
3822.3
3150.9
2632.8
2258.5
2027.6
1785.0
1625.2
310 K
312 K
314 K
308 K
310 K
312 K
314 K
308 K
310 K
312 K
314 K
308 K
310 K
312 K
314 K
58710.0
11105.1
6354.2
4815.2
3868.6
3262.4
2857.5
2545.7
2303.6
2133.3
50746.8
10265.9
6578.6
4881.7
3893.0
3272.0
2844.3
2508.5
2256.5
2048.8
1224.7
801.8
675.4
628.9
585.8
550.1
540.1
536.7
520.9
503.3
1228.0
777.6
684.7
639.3
608.5
575.9
560.5
547.7
533.6
517.1
1186.7
844.4
690.5
621.6
597.2
568.5
560.5
554.0
531.8
526.7
1266.6
882.0
725.5
663.3
621.1
588.4
553.9
534.9
525.5
520.5
29556.5
10776.5
7889.3
6743.5
6010.4
5514.9
5134.9
4854.6
4644.2
4415.4
25226.0
10091.2
7687.4
6593.8
5860.1
5355.9
5047.3
4776.0
4568.5
4407.5
26018.3
10623.3
7791.8
6596.0
5894.0
5393.2
5022.1
4787.9
4581.8
4446.9
28227.2
10002.7
7482.3
6491.0
5785.9
5335.7
4994.3
4775.5
4579.7
4429.9
823.8
506.2
430.8
405.3
378.3
358.8
326.7
305.5
299.8
298.3
765.4
495.4
406.9
368.9
369.4
343.5
317.8
306.3
290.2
282.1
868.8
527.5
435.6
371.8
355.4
337.8
320.9
324.8
309.4
307.2
815.6
509.3
427.1
375.9
348.8
341.2
327.2
323.8
309.3
305.2
112634.2 42848.9
25535.1 9631.3
10619.4 6449.1
6056.7
4973.2
4452.3
4024.0
3796.8
3358.4
3202.9
2881.6
2807.6
2523.1
2509.9
2248.1
2285.2
2016.9
312 K
MM+/ Monte Carlo
6.8
Amber/Monte Carlo
7.4
314 K
11573.7 11747.1 11725.9
7501.0 7244.9 7489.7
5306.3 5088.0 5190.4
4002.1 3772.1 3995.4
3228.8 3028.1 3185.2
2697.7 2517.7 2655.3
2310.3 2137.8 2310.6
2023.6 1901.9 2012.6
1796.8 1689.1 1784.9
1610.9 1524.3 1633.3
OPLS/ Monte Carlo
6.8
308 K
1071.6
730.7
630.4
569.2
527.3
500.6
501.6
498.9
487.8
474.1
310 K
312 K
1100.3 1156.1
715.5
756.5
614.6
651.5
569.9
585.1
544.1
557.7
518.3
526.9
489.5
517.3
488.0
505.7
472.9
496.5
474.1
485.8
OPLS/ Monte Carlo
7.4
314 K
1094.2
744.6
610.2
566.6
535.8
521.5
514.7
503.7
494.7
468.6
PH Effects On 273th Codon Of P53 Gene
85
Table 3. Computed CGT total energy ( kcal/ mol ), belong to AMBER, MM+, BIO+ and OPLS force fields under
four different temperature and 2 various pH values.
Total Energy (Kcal/mol)
Amber/Monte Carlo
6.8
Method
pH condition
time
(PS)
308 K
310 K
312 K
314 K
42395.83 39771.92 30742.54 33686.84
10930.47 9521.98 9001.24 9638.72
6598.47 6153.27 5882.54 6316.91
4910.22 4815.23 4640.13 4913.24
4001.78 3975.48 3855.50 4074.67
3368.65 3382.83 3247.16 3531.85
2960.81 2971.70 2837.54 3089.22
2641.34 2657.94 2519.08 2747.03
2364.04 2408.91 2310.24 2459.74
2164.41 2213.48 2140.25 2268.61
Method
BIO/Monte Carlo
pH condition
6.8
10
20
30
40
50
60
70
80
90
100
time
(PS)
10
20
30
40
50
60
70
80
90
100
308 K
310 K
312 K
MM+/ Monte Carlo
6.8
Amber/Monte Carlo
7.4
314 K
112843.51 43059.53 58922.06 50960.23
25744.46 9842.00 11317.15 10479.31
10828.69 6659.75 6566.27 6791.99
6266.05 5183.86 5027.21 5095.12
4661.60 4234.73 4080.61 4106.44
4006.14 3569.05 3474.48 3485.43
3412.18 3092.25 3069.52 3057.70
3016.90 2733.80 2757.75 2721.93
2719.21 2458.83 2515.61 2469.92
2494.55 2227.63 2345.34 2262.16
308 K
955.79
690.26
590.43
563.78
534.09
514.13
502.07
483.65
471.40
477.48
310 K
312 K
314 K
308 K
310 K
312 K
MM+/ Monte Carlo
7.4
314 K
308 K
981.37
960.40
697.96
699.62
574.51
624.35
530.22
573.58
512.58
548.12
514.11
517.25
502.41
499.34
496.77
488.61
495.29
486.75
478.73
469.58
BIO/Monte Carlo
7.4
1000.10 11915.49 11784.35 11959.10 11939.31
737.49 7610.70 7711.67 7456.99 7703.11
625.93 5249.14 5517.01 5300.08 5403.80
574.36 4031.59 4212.79 3984.11 4208.78
548.84 3360.27 3439.43 3240.12 3398.65
520.73 2842.07 2908.40 2729.76 2868.73
512.44 2467.79 2520.97 2349.84 2523.96
510.17 2236.90 2234.25 2113.97 2225.97
500.31 1994.32 2007.45 1901.19 1998.26
503.66 1834.50 1821.63 1736.35 1846.69
OPLS/ Monte Carlo
6.8
1246.02
905.11
804.79
743.64
701.73
675.08
676.04
673.34
662.26
648.49
308 K
310 K
312 K
314 K
308 K
1399.09
976.22
849.85
803.33
760.25
724.57
714.50
711.11
695.29
677.76
1403.60
953.17
860.25
814.83
784.11
751.48
736.11
723.29
709.13
692.66
1363.36
1021.14
867.17
798.34
773.92
745.20
737.18
730.70
708.46
703.36
1444.39 29765.77 25436.72 26230.32 28440.65
1059.83 10985.77 10301.88 10835.39 10216.09
903.29 8098.60 7898.10 8003.80 7695.71
841.08 6952.81 6804.52 6808.02 6704.35
798.89 6219.72 6070.74 6106.00 5999.28
766.19 5724.22 5566.56 5605.20 5549.11
731.74 5344.17 5257.97 5234.13 5207.72
712.71 5063.88 4986.71 4999.95 4988.89
703.30 4853.53 4779.15 4793.89 4793.11
698.32 4624.72 4618.22 4658.94 4643.34
308 K
310 K
312 K
314 K
Figure 1. Molecular structure of CGT three-nucleotide
998.19
680.60
605.19
579.71
552.70
533.25
501.11
479.96
474.26
472.73
310 K
312 K
1275.82 1332.79
891.08
933.21
790.17
828.23
745.49
761.84
719.70
734.36
693.85
703.59
665.04
693.98
663.57
682.38
648.47
673.22
649.62
662.48
OPLS/ Monte Carlo
7.4
310 K
940.98
670.98
582.44
544.45
544.94
519.12
493.40
481.92
465.81
457.67
312 K
1045.46
704.19
612.29
548.54
532.13
514.48
497.62
501.50
486.14
483.85
314 K
1272.06
922.44
787.98
744.48
713.61
699.31
692.58
681.55
672.53
646.48
314 K
993.43
687.15
604.92
553.75
526.68
519.06
505.06
501.65
487.14
483.06
Journal of Nano Chemical Agriculture , Vol(1) , No(3)
Figure 2. Comparison of minimum energy level in different temperatures in AMBER force field
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