HOMEWORK#8

Sequence analysis / model building

• due on 5/28/98

• David Liu, a student in the department of Lfe Sciences, got a clone from E coli. Here is the DNA sequence:

TGGATGCCATGTTCCGGAGGTAATATGAAGAAATCAATATTATTTATTTTTCTTT

CTGTATTGTCTTTTTCACCTTTCGCTCAGGATGCTAAACCAGTAGAGTCTTCAAA

AGAAAAAATCACACTAGAATCAAAAAAATGTAACATTGCAAAAAAAAGTAATA

AAAGTGGTCCTGAAAGCATGAATAGTAGCAATTACTGCTGTGAATTGTGTTGTA

ATCCTGCTTGTACCGGGTGCTATTAATAATATAAAGGGAACTAAACAGTTCCCT

TTATATTTGTTCTGATTCTGATGATGTCTGTAACGTATGTCCTGTTGCTTTGTTG

AATAAATCGA

Unfortunately, he doesn't know how to use the sequence analysis tools availabled in the internet since he did not take Bioinformatics course before. Could you help him to do the following analysis?

• (1) Find its corresponding polypeptide sequence (DNA -> Protein translation).
• (2) Identify this protein. Is it a new protein?
• (3) Report the total number of negatively charged residues and positively charged residues.
• (4) Color the protein by its charge.
• (5) Draw the hydrophobicity map for this protein using Eisenberg hydrophobicity scale with window size 7. The relative weight of the window edges compared to the window center should set to 40%.
• (6) Please help him to use Prosite scanning tool to find out possible functions or pattern of this protein.
• (7) Calculate its pI and molecular weight.









Answer :

<1>

SEQUENCE : 72 AA; 7909 MW;

MKKSILFIFL SVLSFSPFAQ DAKPVESSKE KITLESKKCN IAKKSNKSGP ESMNSSNYCC ELCCNPACTG CY




<2>

gb|M29255|ECOTOXHS

E.coli heat-stable toxin (st) gene, complete cds. Length = 336

*Plus Strand HSPs:

Score = 1380 (381.3 bits), Expect = 1.0e-104, P = 1.0e-104 Identities = 286/336 (85%),

Positives = 286/336 (85%), Strand = Plus / Plus

*Minus Strand HSPs:

Score = 134 (37.0 bits), Expect = 0.46, P = 0.37 Identities = 30/34 (88%), Positives = 30/34 (88%), Strand = Minus / Plus

*It's a new protein




<3>

Total number of negatively charged residues (Asp + Glu): 6

Total number of positively charged residues (Arg + Lys): 10




<4>

D(Asp), E(Glu)

K(Lys), R(Arg)

MKKSILFIFL SVLSFSPFAQ DAKPVESSKE KITLESKKCN IAKKSNKSGP ESMNSSNYCC ELCCNPACTG CY




<5>

Using the scale Hphob. / Eisenberg et al., the individual values for the 20 amino acids are:

Ala: 0.620 , Arg: -2.530 , Asn: -0.780 , Asp: -0.900

Cys: 0.290 , Gln: -0.850 , Glu: -0.740 , Gly: 0.480

His: -0.400 , Ile: 1.380 , Leu: 1.060 , Lys: -1.500

Met: 0.640 , Phe: 1.190 , Pro: 0.120 , Ser: -0.180

Thr: -0.050 , Trp: 0.810 , Tyr: 0.260 , Val: 1.080

Asx: -0.840 , Glx: -0.795 , Xaa: -0.000

Weights for window positions 1,..,7, using linear weight variation model :

1	2	3	4	5	6	7
0.40	0.60	0.80	1.00	0.80	0.60	0.40
edge			center			edge






<6>

ScanProsite - Protein against PROSITE

MKKSILFIFL SVLSFSPFAQ DAKPVESSKE KITLESKKCN IAKKSNKSGP ESMNSSNYCC ELCCNPACTG CY

[1] PDOC00001 PS00001

ASN_GLYCOSYLATION N - glycosylation site

Number of matches: 2

• 46-49 NKSG
• 54-57 NSSN

-Consensus pattern: N-{P}-[ST]-{P} [N is the glycosylation site]




[2] PDOC00005 PS00005

PKC_PHOSPHO_SITE Protein kinase C - phosphorylation site

Number of matches: 3

• 27-29 SSK
• 36-38 SKK
• 45-47 SNK

-Consensus pattern: [ST]-x-[RK] [S or T is the phosphorylation site]




[3] PDOC00006 PS00006

CK2_PHOSPHO_SITE Casein kinase II - phosphorylation site

Number of matches: 2

• 27-30 SSKE
• 48-51 SGPE

-Consensus pattern: [ST]-x(2)-[DE] [S or T is the phosphorylation site]

-Note: this pattern is found in most of the known physiological substrates.




[4] PDOC00246 PS00273

ENTEROTOXIN_H_STABLE Heat-stable enterotoxins signature

• 59-71 CCELCCNPACTGC

-Consensus pattern: C-C-x(2)-C-C-x-P-A-C-x-G-C [The six C's are involved in disulfide bonds]





<7>

• Molecular weight: 7909.2
• Theoretical pI: 8.72