Peptide Sequence By Using Different Bioinformatics Tools Biology Essay

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Computation of biological data with the help of bioinformatics tools is the cutting edge technology of modern life science. Thus bioinformatics is the computational method of problem solving in life sciences where biological data gathered from different sources are analyzed and redefined by using different data bases.

Expert Protein Analysis system ( ExPASy) is a proteomics server installed in August 1, 1993 by Swiss institute of Bioinformatics. It is the most widely used server for the analysis of protein sequences. The server works on collaboration of Swiss Institute of Bioinformatics with European Bioinformatics Institute. (The ExPASy Molecular biology Server, 2009).

EXPASy server has many tools out of which ProtParam and Motif Scan has been mainly used in this practical. ProtParam is a bioinformatics tool that allows the computational analysis of various physiochemical properties for a protein. Molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index and grand average of hydrophobicity are the parameters that can be computed from ProtParam. (ExPASY Proteomics server, 2009)

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Motif Scan is a proteomics server in which different sequence motifs of a protein are retrieved and analyzed. Sequence motif is actually a pattern of amino acid that is believed to have biological significance on the determination of structure and function of a protein. For example N-glycosylation site motif consists of "Aspargine (N) followed by anything but proline , followed by either serine or Threonine , followed by anything but proline". This pattern is written as N{P}[ST]{P} where N= Asn, P= Pro, S= Ser, T=Thr. Motif scan is actually a tool which scans a peptide sequence against different types of protein profile databases including PROSITE. Secondary structure prediction of a peptide sequence is an attempt to compute that which regions of a polypeptide chain form alpha helices and which region makes beta sheet, without knowing the three dimensional structure. (Lesk AM, 2006)

http://upload.wikimedia.org/wikipedia/en/thumb/c/c4/PBB_Protein_CDKN1B_image.jpg/250px-PBB_Protein_CDKN1B_image.jpg

Fig 1. PDB view of Cyclin-dependent kinase inhibitor 1B (p 27)

Cyclin-dependent kinase inhibitor 1B is a protein in the form of enzyme in human body which is encoded by the CDKN1B gene. (Polyak K et al, 1994) This protein is also known as human p27 and belongs to theCip/Kip family of cyclin dependent kinase (Cdk) inhibitors. The main function of p 27 is to bind with cyclin E-CDK2 or D-CDK4 complexes and prevent their activation. Since E-CDK2 and D-CDK4 are the up regulators of cell cycle, p 27 plays the role of cell cycle inhibitor at G1. This protein can induce cell cycle arrest and serve as tumour suppressors (Chen J et.al, 2009)

The aim of this lab report is to determine the physicochemical characteristics of human p 27 peptide sequences by using Protparam tool of ExPASy followed by determination of potential post-translational modification sites on them. After that Motif scan will be carried out for both wild type and variant sequences of p 27, and finally the secondary structures of human p 27 will also be retrieved and analysed.

AIM

To determine the physiochemical properties of human p 27 peptide sequence and its variant and analyze them

To determine the post translational modification in human p 27 peptide sequence and its variant and analyze them

To perform Motif scan on human p 27 peptide sequence and its variant and analyze them

To predict secondary structure in human p 27 peptide sequence.

MATERIALS AND METHODS

Human p 27 peptide sequence and its variant taken from NCBI (http://www.ncbi.nlm.nih.gov) were entered in Protparam to retrieve their physiochemical properties. Protparam was obtained from http://www.expasy.ch/tools/protparam.html. After that both wild type and variant p 27 peptide sequences were carried out for determination of post translational modification sites by using "NetPhosK" tool to get kinase specific phosphorylation sites. Motif scan was done for human p 27 peptide sequence and its variant by entering these sequences on http://myhits.isb-sib.ch/cgi-bin/motif_scan one by one, and results were analyzed. Finally secondary structure of human p 27 peptide sequence was predicted from Jpred tool of ExPASy and results were analyzed critically.

Result

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Following peptide sequences were obtained from NCBI :

>Human P27

MSNVRVSNGS PSLERMDARQ AEHPKPSACR NLFGPVDHEE LTRDLEKHCR DMEEASQRKW NFDFQNHKPL EGKYEWQEVE KGSLPEFYYR PPRPPKGACK VPAQESQDVS GSRPAAPLIG APANSEDTHL VDPKTDPSDS QTGLAEQCAG IRKRPATDDS STQNKRANRT EENVSDGSPN AGSVEQTPKK PGLRRRQT

Figure 2. Human p 27 peptide sequence, wild type (NCBI , 2009)

>Variant human p27

MSNVRVSNGS PSLERMDARQ AEHPKPSACR NLFGPVDHEE LTRDLEKHCR DMEEASQRKW

NFDFQNHKPL EGKYEWQEVE KGSLPEFYYR PPRPPKGACK VPAQESQDGS GSRPAAPLIG

APANSEDTHL VDPKTDPSDS QTGLAEQCAG IRKRPATD

Figure 3. Variant of Human p 27 sequence (NCBI 2009)

ProtParam Analysis OF HUMAN p27, WILD type and variant peptide sequences

When the human p 27, wild type and variant peptide sequences obtained from NCBI were entered in Protparam, following results were obtained:

10 20 30 40 50 60

MSNVRVSNGS PSLERMDARQ AEHPKPSACR NLFGPVDHEE LTRDLEKHCR DMEEASQRKW

70 80 90 100 110 120

NFDFQNHKPL EGKYEWQEVE KGSLPEFYYR PPRPPKGACK VPAQESQDVS GSRPAAPLIG

130 140 150 160 170 180

APANSEDTHL VDPKTDPSDS QTGLAEQCAG IRKRPATDDS STQNKRANRT EENVSDGSPN

190

AGSVEQTPKK PGLRRRQT

Fig. 4 Protparam view of human p 27, wild type

10 20 30 40 50 60

MSNVRVSNGS PSLERMDARQ AEHPKPSACR NLFGPVDHEE LTRDLEKHCR DMEEASQRKW

70 80 90 100 110 120

NFDFQNHKPL EGKYEWQEVE KGSLPEFYYR PPRPPKGACK VPAQESQDGS GSRPAAPLIG

130 140 150

APANSEDTHL VDPKTDPSDS QTGLAEQCAG IRKRPATD

Fig 5. Protparam view of human p 27 variant sequence

The physiochemical properties of wild type and variant sequence of human p 27 obtained from ProtParam analysis has been tabulated in table 1 below:

Table 1. Comparison of Physiochemical Properties of human p27, wild type and variant peptide sequences

SN

Properties

Wild type p27 , (Human)

Variant p 27,

(Human)

Remarks

1

Number of amino acids

198

158

2

Molecular weight

22073.2

17651.5

3

Theoretical PI

6.54

5.77

4

Total no. of negatively charged residues (Asp + Glu)

31

26

5

Total no. of positively charged residues (Arg + Lys)

30

22

6

Molecular Formula

C935H1483N295O312S7

C756H1180N230O246S7

7

Total number of atoms

3032

2419

8

Extinction coefficients at 280 nm measured in water ( assuming all Cys residues appear as half Cystines)

15720

15720

(unit M-1 cm-1)

9

Extinction coefficients at 280 nm measured in water ( assuming no Cys residues appear as half Cystines)

15470

15470

(unit M-1 cm-1 )

10

Estimated half life

30 hrs (mammalian reticulocytes, in vitro), >20 hrs (yeast, in vivo), >10 hrs (E. Coli ,in vivo

30 hrs (mammalian reticulocytes, in vitro), >20 hrs (yeast, in vivo), >10 hrs (E. Coli ,in vivo

11

Instability index

66.05

58.23

Indicates that protein is unstable

12

Aliphatic index

44.39

46.39

13

Grand average of hydropathicity (Gravity)

-1.261

-1.146

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DETERMINATION OF POST TRANSLATIONAL MODIFICATION

(from NetPhosK TOOL)

Following Kinase specific phosphorylation sites on wild type and variant of human p27 were detected from "Net PhosK" tool

Human P 27 wild type

Site Kinase Score

---------------------

S-2 cdc2 0.51

S-7 PKA 0.63

S-10 p38MAPK 0.55

S-10 GSK3 0.51

S-10 cdk5 0.51

T-42 CKII 0.63

S-56 DNAPK 0.59

S-83 CKII 0.53

S-83 PKA 0.61

S-83 cdc2 0.53

S-106 DNAPK 0.63

S-112 PKC 0.51

S-125 CKII 0.54

S-138 PKA 0.50

S-138 PKG 0.54

S-140 DNAPK 0.58

S-140 ATM 0.51

T-142 CKII 0.54

T-142 PKC 0.50

T-157 PKB 0.82

T-157 PKC 0.51

T-162 DNAPK 0.61

T-187 PKC 0.53

T-187 cdk5 0.76

T-198 RSK 0.64

T-198 PKC 0.54

T-198 PKA 0.82

---------------------

Highest Score: 0.82 PKB at position 157

Fig 6. NetPhosK method of determination of kinase specific phosphorylation sites on human p 27 (wild type) without ESS filtering

Human p 27, variant

Site Kinase Score

---------------------

S-2 cdc2 0.51

S-7 PKA 0.63

S-10 p38MAPK 0.55

S-10 GSK3 0.51

S-10 cdk5 0.51

T-42 CKII 0.63

S-56 DNAPK 0.59

S-83 CKII 0.53

S-83 PKA 0.61

S-83 cdc2 0.53

S-106 DNAPK 0.63

S-125 CKII 0.54

S-138 PKA 0.50

S-138 PKG 0.54

S-140 DNAPK 0.58

S-140 ATM 0.51

T-142 CKII 0.54

T-142 PKC 0.50

T-157 PKB 0.77

---------------------

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Highest Score: 0.77 PKB at position 157

Fig 7. NetPhosK method of determination of kinase specific phosphorylation sites on human p 27 (variant) without ESS filtering

http://www.cbs.dtu.dk/services/NetPhosK/tmp/916951/prediction.gif

Fig . 8. NetPhosK method of determination of kinase specific phosphorylation sites on human p 27 (wild type) with Kinase Landscape

http://www.cbs.dtu.dk/services/NetPhosK/tmp/101610/prediction.gif

Fig 9. NetPhosK method of determination of kinase specific phosphorylation sites on human p 27 (variant) with kinase landscape

RESULT OF MOTIF SCAN OF HUMAN p 27 (WILD TYPE SEQUENCE)

Following results were obtained from Motif scan analysis of human p 27 peptide sequence (wild type):

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Motif Scan Results

user: GUEST

Query Protein

temporarily stored here.

Database of motifs

PROSITE patterns [pat], PROSITE patterns (frequent match producers) [freq_pat], PROSITE profiles [prf], HAMAP profiles [hamap], More profiles [pre], Pfam HMMs (local models) [pfam_fs], Pfam HMMs (global models) [pfam_ls], PeroxiBase profiles [perox].

Reference

Hulo N, Bairoch A, Bulliard V, Cerutti L, Cuche BA, de Castro E, Lachaize C, Langendijk-Genevaux PS, Sigrist CJ.

The 20 years of PROSITE.

Nucleic Acids Res. 2008; 36(Database issue):D245-9.

searching PeroxiBase profiles

searching HAMAP profiles

searching PROSITE patterns

searching PROSITE patterns (frequent match producers)

searching PROSITE profiles

searching More profiles

searching Pfam HMMs (local models)

searching Pfam HMMs (global models)

postprocessing

Summary

Original output

perox, hamap, pat, freq_pat, prf, pre, pfam_fs, pfam_ls.

Matches map

(features from query are above the ruler, matches of the motif scan are below the ruler)

match map segment

match map segment

match map segment

match map segment

Legends: 1, freq_pat:ASN_GLYCOSYLATION [?]; 2, freq_pat:CAMP_PHOSPHO_SITE [?]; 3, freq_pat:CK2_PHOSPHO_SITE [?]; 4, freq_pat:MYRISTYL [?]; 5, freq_pat:PKC_PHOSPHO_SITE [?]; 6, freq_pat:TYR_PHOSPHO_SITE [?]; 7, prf:NLS_BP [?]; 8, pre:NLS_BP [?]; 9, pfam_ls:OGFr_III [?].

List of matches

FT MYHIT 168 171 freq_pat:ASN_GLYCOSYLATION [?]

FT MYHIT 173 176 freq_pat:ASN_GLYCOSYLATION [?]

FT MYHIT 195 198 freq_pat:CAMP_PHOSPHO_SITE [?]

FT MYHIT 83 86 freq_pat:CK2_PHOSPHO_SITE [?]

FT MYHIT 111 116 freq_pat:MYRISTYL [?]

FT MYHIT 120 125 freq_pat:MYRISTYL [?]

FT MYHIT 177 182 freq_pat:MYRISTYL [?]

FT MYHIT 56 58 freq_pat:PKC_PHOSPHO_SITE [?]

FT MYHIT 187 189 freq_pat:PKC_PHOSPHO_SITE [?]

FT MYHIT 68 74 freq_pat:TYR_PHOSPHO_SITE [?]

FT MYHIT 153 169 prf:NLS_BP [?]

FT MYHIT 153 170 pre:NLS_BP [?]

FT MYHIT 30 80 pfam_fs:CDI [!]

FT MYHIT 30 80 pfam_ls:CDI [!]

FT MYHIT 105 124 pfam_ls:OGFr_III [?]

Match details

match detail

match score

motif information

match map segment

Status: ?

pos.: 168-171

freq_pat:ASN_GLYCOSYLATION

N-glycosylation site.

[ entry ]

Legends: 1, carbohydrate.

match map segment

Status: ?

pos.: 173-176

^ image ^

match map segment

Status: ?

pos.: 195-198

freq_pat:CAMP_PHOSPHO_SITE

cAMP- and cGMP-dependent protein kinase phosphorylation site.

[ entry ]

Legends: 1, phosphorylation.

^ image ^

match map segment

Status: ?

pos.: 83-86

freq_pat:CK2_PHOSPHO_SITE

Casein kinase II phosphorylation site.

[ entry ]

Legends: 1, phosphorylation.

^ image ^

match map segment

Status: ?

pos.: 111-116

freq_pat:MYRISTYL

N-myristoylation site.

[ entry ]

Legends: 1, myristyl.

match map segment

Status: ?

pos.: 120-125

match map segment

Status: ?

pos.: 177-182

^ image ^

match map segment

Status: ?

pos.: 56-58

freq_pat:PKC_PHOSPHO_SITE

Protein kinase C phosphorylation site.

[ entry ]

Legends: 1, phosphorylation.

match map segment

Status: ?

pos.: 187-189

^ image ^

match map segment

Status: ?

pos.: 68-74

freq_pat:TYR_PHOSPHO_SITE

Tyrosine kinase phosphorylation site.

[ entry ]

Legends: 1, phosphorylation.

^ image ^

match map segment

match map segment

match map segment

match map segment

Status: ?

pos.: 153-169

raw-score = 3

N-score = 3.000

E-value = 2.1e+04

prf:NLS_BP

Bipartite nuclear localization signal profile.

[ entry ]

[ graphics ]

^ image ^

match map segment

match map segment

match map segment

match map segment

Status: ?

pos.: 153-170

raw-score = 3

N-score = 3.000

E-value = 2.1e+04

pre:NLS_BP

Bipartite nuclear localization signal.

[ entry ]

[ graphics ]

^ image ^

match map segment

match map segment

match map segment

match map segment

Status: !

pos.: 30-80

raw-score = 98.0

N-score = 34.469

E-value = 7.2e-28

pfam_fs:CDI

Cyclin-dependent kinase inhibitor

[ entry ]

^ image ^

match map segment

match map segment

match map segment

match map segment

Status: !

pos.: 30-80

raw-score = 99.9

N-score = 35.025

E-value = 2e-28

pfam_ls:CDI

Cyclin-dependent kinase inhibitor

[ entry ]

^ image ^

match map segment

match map segment

match map segment

match map segment

Status: ?

pos.: 105-124

raw-score = 0.3

N-score = 7.047

E-value = 1.9

pfam_ls:OGFr_III

Opioid growth factor receptor repeat

[ entry ]

^ image ^

Fig 10. Result of Motif scan of human p 27 peptide sequence, wild type (Motif Scan , 2009)

RESULT OF MOTIF SCAN OF HUMAN p 27 (VARIANT SEQUENCE)

Summary

Original output

perox, hamap, pat, freq_pat, prf, pre, pfam_fs, pfam_ls.

Matches map

(features from query are above the ruler, matches of the motif scan are below the ruler)

match map segment

match map segment

match map segment

match map segment

Legends: 1, freq_pat:CK2_PHOSPHO_SITE [?]; 2, freq_pat:MYRISTYL [?]; 3, freq_pat:PKC_PHOSPHO_SITE [?]; 4, freq_pat:TYR_PHOSPHO_SITE [?].

List of matches

FT MYHIT 83 86 freq_pat:CK2_PHOSPHO_SITE [?]

FT MYHIT 111 116 freq_pat:MYRISTYL [?]

FT MYHIT 120 125 freq_pat:MYRISTYL [?]

FT MYHIT 56 58 freq_pat:PKC_PHOSPHO_SITE [?]

FT MYHIT 68 74 freq_pat:TYR_PHOSPHO_SITE [?]

FT MYHIT 30 80 pfam_fs:CDI [!]

FT MYHIT 30 80 pfam_ls:CDI [!]

Match details

match detail

match score

motif information

match map segment

Status: ?

pos.: 83-86

freq_pat:CK2_PHOSPHO_SITE

Casein kinase II phosphorylation site. 

[ entry ]

Legends: 1, phosphorylation.

^ image ^ 

match map segment

Status: ?

pos.: 111-116

freq_pat:MYRISTYL

N-myristoylation site. 

[ entry ]

Legends: 1, myristyl.

match map segment

Status: ?

pos.: 120-125

^ image ^ 

match map segment

Status: ?

pos.: 56-58

freq_pat:PKC_PHOSPHO_SITE

Protein kinase C phosphorylation site. 

[ entry ]

Legends: 1, phosphorylation.

^ image ^ 

match map segment

Status: ?

pos.: 68-74

freq_pat:TYR_PHOSPHO_SITE

Tyrosine kinase phosphorylation site. 

[ entry ]

Legends: 1, phosphorylation.

^ image ^ 

match map segment

match map segment

match map segment

match map segment

Status: !

pos.: 30-80

raw-score =98.0

N-score =34.469

E-value = 7.2e-28

pfam_fs:CDI

Cyclin-dependent kinase inhibitor

[ entry ]

^ image ^ 

match map segment

match map segment

match map segment

match map segment

Status: !

pos.: 30-80

raw-score =99.9

N-score =35.025

E-value = 2e-28

pfam_ls:CDI

Cyclin-dependent kinase inhibitor

[ entry ]

^ image ^ 

Fig 11. Result of Motif scan of human p 27 variant sequence (Motif Scan , 2009)

SECONDARY STRUCTURE PREDICTION

Following secondary structure of human p 27 was predicted when the sequence was entered in "Jpred" tool of ExPASy:

(H= helix, E=Extended strand, - means others)

Fig 12. Predicted secondary structure of human p 27 peptide sequence from Jpred

Jpred is a consensus method of protein secondary structure prediction at University of Dundee. This metod was used to predict the secondary structure in human p 27 peptide sequence. The out put of the experiment is on figure 12, where blue colour shows complete identity at a position. Shades of red colour shows the conservation profile of chemical properties. The more red a position is, the higher the level of conservation of chemical properties of the amino acids. "Jnet" in Fig 12 is final secondary structure prediction for query. Regarding coiled coil prediction, "-" indicates less than 50% probability, c indicates 50% to 90% probability, and C indicates more than 90% probability. Similarly, Jnet_25, Jnet_5, and Jnet_0 are the prediction of burial with <25% solvent accessibility, <5% exposure, and 0% exposure. The value of Jnet reliability of prediction accuracy ranges from 0 to 9.

Discussion

Human p27 (wild type) is a 198 aminoacid peptide sequence with molecular weight 22073.2. Its variant p 27 (kip 1) consists of 158 aminoacids and consequently bears relatively less molecular weight i.e. 17651.5. As we know, the bigger a peptide sequence, the less its stability is. Data shows that instability index (II) of wild type p 27 is 66.05 while that of variant is 58.23. Instability index (II) is actually the measure of protein that determines that whether the protein is stable in test tube or not. If the value of instability index is less than 40, the protein is supposed to be stable in testube.Otherwise, the protein is considered to be unstable. (Guruprasad K, Reddy BV, Pandit MW, 1990). Since the instability index values of both the peptide sequences are more than 40, they are unstable in vitro, however the wild type p 27 has been found to be more unstable than its variant, probability due to it bigger size.

The aliphatic index of the wild type human p 27 is 44.39, while that of variant is 46.39. It indicates that volume occupied by the side chains of variant peptide is slightly larger than that occupied by the side chains of wild type.

Post translational modification determination done to predict the kinase specific phosphorylation sites shows that highest score of Protein Kinase B (PKB) was seen in 157 amino acid position of both wild type and variant p27. However the score value of wild type was higher ( 0.82) than that of variant (0.77). Almost all the phosphorylation in p27 is seen at serine and threonine residue. This fact has been supported by the results of post translational modification at Fig 6, 7, 8 and 9.

Motif scan result shows that three cyclin dependent kinase inhibitor motifs with three sequences were seen in both wild type and variant p 27 sequences. Motif is actually the functional unit of a sequence. The major function of human p 27 is cell cycle arrest at G1. A growth factor dependent nuclear kinase phosphorylates p 27 and regulates cell cycle progression. Actually the degree of phosphorylation on p27 is inversely proportional to the cell cycle inhibition activity of p27. Protein Kinase B mediated phosphorylation of p 27 leads to inhibition its activity and may result in breast carcinoma. Similarly, Akt dependent phosphorylation of T 198 residue of p27, may also lead to breast carcinoma. The another cause of breast carcinoma might be mutation at T 157 residue of p 27. Apart from breast carcinoma, mutation on p 27, also results in multiple endocrine neoplasia.

Human p 27 is expressed in almost all the tissues of human body, mostly in the liver and kidney. It is predominantly found in quiescent cells. In a cell, it is present at the nucleus at its active state. Phosphorylation at serine 10 leads to translocation of p 27 to cytoplasm and inactivation.

The above scenario indicates that there is profound role of physio chemical properties, and post translational modification on the over all function of a protein. These data show that the performance of p 27 has been determined by the different phosphorylations occurring at its different aminoacid residues. Different mutations leading to different carcinomas are also determined by such post translational modifications.

CONCLUSION

This experiment leads to a conclusion that function of a protein has been determined by its physiochemical properties and post translational modification. Hence these are the determinat factor for the over all activity of the protein. Any alteration on these determinants might lead to some terrible pathogenic conditions like carcinoma.

.