HOMEWORK#8

Sequence analysis / model building

* due on 5/28

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* David Liu, same guy from last homework, also isolated a cDNA clone from

snake venom library. The nucleotide sequence of this cDNA clone is

shown here:

AAAACCATCAAATATGTTATGCTGGAATGCAACGAACTGATCCCGCTGTTC

TACGAAACCTGCCCGGCTGGTGAAAACATCTGCTACGAAATGTTCATGGTT

GCTACCCCGAAAGTTCCGTGCGAACGTGGTTGCATCGACGTTTGCCCGGAA

TCTTCTCTGATCGTTAAATACGTTTGCTGCAACACCGACCGTTGCCAGTAAT

CCAGCGCCTGATCTCTCGAAATAAAAGCCGCATTG

1) Please help him to find its corresponding polypeptide

sequence (DNA -> Protein translation).

Using the translation tool of Expasy, I think this is the most possible sequence:

MLECNELIPL FYETCPAGEN ICYEMFMVAT PKVPCERGCI DVCPESSLIV KYVCCNTDRCQ

2) Please help him to calculate pI/Mw of this polypeptide,

perform the trypsin cutting , analyze the cutting pattern and

report the fragments with molecular weight over 500 Dalton.

Molecular weight: 6917.13

Theoretical pI: 4.29

The selected enzyme is: Trypsin

All cysteines in reduced form.

Methionines have not been oxidized.

Displaying peptides with a mass bigger than 500 Dalton.

Using monoisotopic masses of the occurring amino acid residues and giving

peptide masses as [M+H]+.

mass position peptide sequence

3699.66 1- 32 MLECNELIPLFYETCPAGENICYEMFMVATPK

1462.73 38- 51 GCIDVCPESSLIVK

973.39 52- 59 YVCCNTDR

603.29 33- 37 VPCER

(3) Please help him to identify this toxin. Is it a new

toxin?

In my opinion, this protein seems to be a new one.

(I put the sequence on line and check similarities. The best result I got were showing below:

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>sp|Q02454|CX1_NAJSP (CTX) CARDIOTOXIN PRECURSOR.

Length = 81

Score = 296 (104.2 bits), Expect = 5.1e-27, P = 5.1e-27

Identities = 48/59 (81%), Positives = 58/59 (98%)

Query: 2 LECNELIPLFYETCPAGENICYEMFMVATPKVPCERGCIDVCPESSLIVKYVCCNTDRC 60

L+CN+L+PLFY+TCPAG+N+CY+MFMVATPKVP +RGCIDVCP+SSL+VKYVCCNTDRC

Sbjct: 22 LKCNKLVPLFYKTCPAGKNLCYKMFMVATPKVPVKRGCIDVCPKSSLLVKYVCCNTDRC 80

>sp|P01444|CX3_NAJAT CYTOTOXIN 3 PRECURSOR (CARDIOTOXIN ANALOGUE III) (CTX III)

(CARDIOTOXIN C-10) (CYTOTOXIN IV).

Length = 81

Score = 296 (104.2 bits), Expect = 5.1e-27, P = 5.1e-27

Identities = 48/59 (81%), Positives = 58/59 (98%)

Query: 2 LECNELIPLFYETCPAGENICYEMFMVATPKVPCERGCIDVCPESSLIVKYVCCNTDRC 60

L+CN+L+PLFY+TCPAG+N+CY+MFMVATPKVP +RGCIDVCP+SSL+VKYVCCNTDRC

Sbjct: 22 LKCNKLVPLFYKTCPAGKNLCYKMFMVATPKVPVKRGCIDVCPKSSLLVKYVCCNTDRC 80

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Since the identities were both only 81%, I regarded it to be a new protein! )

(4) Please help him to use Prosite scanning tool to find out

possible functions or pattern of this polypeptide.

The Snake toxins signature 38-58 GCIDVCPESSLIVKYVCCNTD

(5) David would like to see its structure. Could you help him

to find structure of this toxin or make a model if it is a

new protein? Show structure on your homwpage ( 3 different

views).

This a structure I got from Swiss-Expasy (viewed from 3 different directions):