Download 3D protein structure

Assignment
To find the mutation being introduced into the p53 protein if the primer sequence provided
was used to run the PCR (note: the primer might be either a forward or a reverse sequence)
PRIMER SEQUENCE
STEP 1: Obtain the p53 nucleotide sequence from PUBMED database.
Go to the PUBMED database and select ‘Nucleotide’ in the dropdown menu
Type in p53 in the search box and click search.
Select Homo Sapiens in the ‘Results by taxon’ section. Select the first p53 mRNA sequence
shown (Note: this is a complete coding sequence for the p53 protein), 2,451 bp linear mRNA,
Accession: AB082923.1, GI: 23491728
At the very end of the page, you will see the nucleotide sequence of the p53 protein.
Immediately above the sequence, there is the amino acid sequence under the subheading
‘CDS’. Click CDS to highlight the coding region in the nucleotide sequence.
Now copy the nucleotide sequence into the space below and highlight the CDS region in red:
STEP 2: Compare and find out which region your particular primer binds to in the p53 coding
sequence.
Using google, search for ‘nucleotide blast’ – an NCBI sequence alignment program and open
the following page.
Copy and paste your primer sequence in the QUERY box and your p53 nucleotide sequence
in the SUBJECT box. Click on BLAST.
The colourful box shows a visual representation of your results while the exact nucleotide
matches are shown below.
The query sequence (1-29 nucleotides in length) matched exactly to the subject sequence
from its nucleotide sequence starting from 194-222. Now go back to your nucleotide
sequence and highlight in yellow, the corresponding region (194-222). NOTE: There will be a
mismatch of one nucleotide (which is your mutation). Also, the reverse primers will map in
the 3’ to 5’ direction.
STEP 3: Find out which amino acid is being mutated and what the mutation is.
Copy your CDS (coding sequence highlighted in red) from the nucleotide sequence below.
A codon is a group of three nucleotides coding for a single amino acid. Please refer to the
codon table provided below to know the composition of each amino acid. As you will notice,
ATG is a start codon (first codon of your sequence in red) and TGA is a stop codon (last codon
of your sequence in red).
Amino
acid
Ala/A
Codons
GCT, GCC, GCA,
GCG
CODON TABLE
Compressed
Amino
acid
GCN
Leu/L
Codons
TTA, TTG, CTT,
CTC, CTA, CTG
YTR, CTN
AAR
Arg/R
CGT, CGC, CGA,
CGG, AGA, AGG
CGN, MGR
Lys/K
AAA, AAG
Asn/N
Asp/D
Cys/C
AAT, AAC
GAT, GAC
TGT, TGC
AAY
GAY
TGY
Met/M
Phe/F
Pro/P
Gln/Q
CAA, CAG
CAR
Ser/S
ATG
TTT, TTC
CCT, CCC, CCA,
CCG
TCT, TCC, TCA,
TCG, AGT, AGC
Glu/E
GAA, GAG
GAR
Thr/T
Gly/G
GGT, GGC, GGA,
GGG
CAT, CAC
ATT, ATC, ATA
GGN
Trp/W
CAY
ATH
Tyr/Y
Val/V
His/H
Ile/I
START
ATG
(adapted from WIKIPEDIA)
STOP
Compressed
TTY
CCN
TCN, AGY
ACT, ACC, ACA,
ACG
TGG
ACN
TAT, TAC
GTT, GTC, GTA,
GTG
TAA, TGA, TAG
TAY
GTN
TAR, TRA
Next, split your above sequence into groups of three (i.e. into each codon) as shown below.
1
2
3
4
5
6
7
8
9
10
11
12 13
14 15
16
17
18
19
Now, copy and paste below the highlighted primer region and pick out the mutation site and
fill it in the box given below.
Primer sequence in codons:
is the codon being mutated.
Using your codon table, identify which amino acid the above codon (in the box) codes for and
write it below.
codes for the following amino acid ___________________________ .
From your primer sequence, identify the nucleotide that has been mutated.
Has been mutated to
which means that the amino acid
has been mutated to
.
Please fill out the following sentence as your result from this exercise:
In the ______ amino acid position, the codon ____________ has been mutated to
__________ which means that the amino acid ________________________ has been
changed to _________________________ . This can be represented using the notation
______________________ .
EXERCISE
A protein’s secondary structures may be visualized in 3D using various softwares like
DeepView. These structures are generated using different techniques, for e.g. x-ray
crystallography and published in various journals. All these structures are also available in
databases like RCSB protein data bank (see below). In this exercise, we shall look into the p53
protein structure and observe the mutations being introduced in this project.
I have previously downloaded the p53 homotetramer structure, PDB ID: A2HI. Open this
structure using the DeepView software. We can see the various control and visualization
panels below:
We can un-highlight a single p53 protein using the control panel (right).
Now, we can highlight a particular mutation, for e.g. R248G which is a change from Arginine
to Glycine at the 248th position, using the control panel. In this structure we can observe
Arginine at the 248th position. In the PCR we did yesterday, one set of primers introduced a
change in this particular Arginine and changed it into a Glycine.
Likewise, you may also study how this would affect various changes like DNA binding effects
(if the mutation is in the DBD domain), electrostatic potentials, creation and deletion of
different types of bonds, etc using this software. A pdf manual for this software is available
for your perusal in your downtime. You may also find other p53 structures with other
mutations in the RCSB database. Some examples are below:
1) V143A - http://www.rcsb.org/pdb/explore.do?structureId=2j1w
PDB ID code 2J1W
2) R248G - http://www.nature.com/onc/journal/v26/n15/fig_tab/1210291f1.html#figure-title
PDB ID code 2AHI
3) R175H - http://www.nature.com/onc/journal/v26/n15/fig_tab/1210291f1.html#figure-title
PDB ID code 2AHI