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).

5'3' Frame 1

K T I K Y V Met L E C N E L I P L F Y E T C P A G E N I C Y E Met F Met V A T P K V P C E R G C I D V C P E S S L I V K Y V C C N T D R C Q Stop S S A Stop S L E I K A A L

(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.

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]+.

The peptide masses from your sequence are:

[Theoretical pI/Mw: 4.29 / 6917.13 ]

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?

Identities = 48/59 (81%), so it is a new toxin

(4) Please help him to use Prosite scanning tool to find out possible functions or pattern of this polypeptide.

**************************

* Snake toxins signature *

**************************

Snake toxins belong to a family of proteins [1,2,3] which groups short and

long neurotoxins, cytotoxins and short toxins, as well as a other miscellanous venom

peptides.

Most of these toxins act by binding to the nicotinic acetylcholine receptors in

the postsynaptic membrane of skeletal muscles and prevent the binding of

acetylcholine, thereby blocking the excitation of muscles.Snake toxins are

proteins that consist of sixty to seventy five amino acids. Among the invariant

residues are eight cysteines all involved in disulfide bonds. A signature pattern

was developed [4] which includes four of these cysteines as well as a conserved

proline thought to be important for the maintenance of the tertiary structure.

The second cysteine in the pattern is linked to the third one by a disulfide bond.

-Consensus pattern: G-C-x(1,3)-C-P-x(8,10)-C-C-x(2)-[PDEN]

[The four C's are involved in disulfide bonds]

-Sequences known to belong to this class detected by the pattern: Most of the

snake toxins are detected except for fasciatoxin, which is an atypical short

neurotoxin, and eight toxins which have a very low activity.

-Other sequence(s) detected in SWISS-PROT: 13.

-Last update: November 1995 / Pattern and text revised.

(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).