HOMEWORK#8

• 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).

(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.

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

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

(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).

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(1).Ans :

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

(2).Ans :

Theoretical pI/Mw: 4.29 / 6917.13

MS-Digest Results :

Digestion enzyme: Trypsin
Cysteines modified by: unmodified

Start	End	MH+(mi)	MH+(av)	Sequence
33	37	603.2925	603.7	(K)VPCER(G)
52	59	973.3872	974.1	(K)YVCCNTDR(C)
52	61	1204.4549	1205.4	(K)YVCCNTDRCQ(-)
38	51	1462.7286	1463.8	(R)GCIDVCPESSLIVK(Y)
33	51	2047.0026	2048.5	(K)VPCERGCIDVCPESSLIVK(Y)
38	59	2417.0973	2418.8	(R)GCIDVCPESSLIVKYVCCNTDR(C)
38	61	2648.1651	2650.1	(R)GCIDVCPESSLIVKYVCCNTDRCQ(-)
33	59	3001.3714	3003.5	(K)VPCERGCIDVCPESSLIVKYVCCNTDR(C)
1	32	3699.6627	3702.4	(-)MLECNELIPLFYETCPAGENICYEMFMVATPK(V)
1	37	4283.9368	4287.1	(-)MLECNELIPLFYETCPAGENICYEMFMVATPKVPCER(G)
1	51	5727.6470	5731.9	(-)MLECNELIPLFYETCPAGENICYEMFMVATPKVPCERGCIDVCPESSLIVK(Y)

(3).Ans :

Yes.It is a new toxin.
THe nearest one is :CARDIOTOXIN PRECURSOR pir
Score = 296 (142.8 bits)     
Identities = 48/59 (81%)

Query:     2 LECNELIPLFYETCPAGENICYEMFMVATPKVPCERGCIDVCPESSLIVKYVCCNTDRC 60            
             L+CN+L+PLFY+TCPAG+N+CY+MFMVATPKVP +RGCIDVCP+SSL+VKYVCCNTDRC
Sbjct:    22 LKCNKLVPLFYKTCPAGKNLCYKMFMVATPKVPVKRGCIDVCPKSSLLVKYVCCNTDRC 80

(4).Ans :

ScanProsite - Protein against PROSITE

1. PDOC00005 PS00005  PKC_PHOSPHO_SITE
   Protein kinase C phosphorylation site

        Number of matches: 2
        1      30-32 TPK                                                              
        2      57-59 TDR 
---------------------------------------------

2. PDOC00245 PS00272  SNAKE_TOXIN
   Snake toxins signature

        38-58 GCIDVCPESSLIVKYVCCNTD

                                                        
(5).Ans :