assignment of prosite motifs to topological regions: application to a novel database of well...
TRANSCRIPT
Assignment of PROSITE motifs to topological regions: Application to a novel database of well characterised
transmembrane proteins
Tim Nugent
6 Month Report 11-05-07
Identification of Transmembrane Regions
Most Hydrophobic: Least Hydrophobic:
4.5 Arginine -4.54.2 Lysine -3.93.8 -3.5
Phenylalanine 2.8 -3.52.5 -3.5
IsoleucineValineLeucine Glutamic acid
AsparagineCysteine Glutamine
To generate data for a plot, the protein sequence is scanned with a moving window of size 19-21 residues. At each position, the mean hydrophobic index of the amino acids within the window is calculated and that value plotted as the midpoint of the window.
Aquaporin
The Positive Inside Rule
Hydrophobic Positive
0
0.1
0.2
0.3
0.4
0.5
0.6
Inside Loops
Outside Loops
Helices
Ave
rag
e a
min
o a
cid
fra
ctio
nHydrophobic: Val, Phe, Ile, Leu, Met. Positive: Lys, Arg, His.
Cytoplasmic loops are enriched in positively charged residues: the 'positive-inside rule' of von Heijne
Assembling a novel data set of transmembrane proteins
In order to study and predict features of transmembrane proteins, the use of a high quality data set containing sequences with experimentally confirmed TM regions is essential.
The data set was based on the widely used Möller test set (2001).
Additional data was collected from MPTOPO, OPM, SWISSPROT and from the literature.
Sequences were blasted against the PDB in order to identify entries for which the TM region had complete structural coverage.
This set was then homology reduced at the 40% sequence identity level.
The makeup of the final data set contains 141 sequences, all with available structures, verifiable topology and N-terminal locations.
111 Alpha-helical proteins
30 Beta-barrel proteins
Column Example Entry
swissprotaccessionpdbn_terminal_locationn_terminal_in_outmembrane_typehelix_counttopologyswissprot_sequencesequence_lengthclassdatasourcedescriptiontaxonomydomainid
ANASPQ8YSC41XIO:APeriplasmicOutsideBacterial gram-negative inner membrane7A.3,26;B.35,56;C.70,89;D.99,121;E.127,148;F.168,185;G.195,218MNLESLLHWIYVAGMTIGALHFWSLSRNPRGVPQYEYLVAMFI...261Alpha-helicalOPMBacteriorhodopsin - Anabaena sp. (strain PCC 7120)Bacteria; Cyanobacteria; Nostocales; Nostocaceae; Nostoc.Prokaryotes1052
MySQL table schema
Hydrophobic Positive Negative Aromatic Polar0
0.1
0.2
0.3
0.4
0.5
0.6
Aver
age
amin
o ac
id fr
actio
n
Hydrophobic: Val, Phe, Ile, Leu, Met. Positive: Lys, Arg, His. Negative: Asp, Glu. Aromatic: Phe, Trp, Tyr. Polar: Cys, Pro, His, Asn, Gln, Ser, Thr.
Amino acid composition
Hydrophobic Positive Negative Aromatic Polar
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Inside Loops
Outside Loops
Helices
Aver
age
amin
o ac
id fr
actio
n
Beta-barrel proteins Alpha-helical proteins
Assignment of PROSITE motifs to topological regions
We next explored the possibility that motifs from the PROSITE database could be used as constraints in subsequent topology prediction steps, by identifying a bias in their inside/outside frequency.
Extracelullar
Cytoplasm
Prosite ID Description Inside Outside Helix Multiple n χ2
PS00008 N-myristoylation site 20.23% 32.11% 44.82% 2.68% 598 7.89
PS00006 Casein kinase II phosphorylation site 53.97% 38.41% 5.96% 1.66% 302 18.83
PS00005 Protein kinase c phosphorylation site 63.08% 29.23% 7.69% 0.00% 260 50.23
PS00001 N-glycosylation site 49.51% 33.01% 13.59% 3.88% 103 5.32
PS00004 cAMP- and cGMP-dependent protein kinase phosphorylation site 67.86% 28.57% 3.57% 0.00% 28 10.72
PS00009 Amidation site 85.71% 7.14% 0.00% 7.14% 14 7.65
PS00029 Leucine Zipper 27.27% 0.00% 27.27% 45.45% 11 3.47
PS00221 MIP family signature 100.00% 0.00% 0.00% 0.00% 4 4.63
PS50857 Cytochrome c oxidase subunit II signature 0.00% 100.00% 0.00% 0.00% 4 3.46
Alpha-helical protein PROSITE motif assignments
Prosite ID Description Inside Outside Beta-sheet Multiple n χ2
PS00008 N-myristoylation site 30.29% 34.58% 29.49% 5.09% 373 24.88
PS00006 Casein kinase II phosphorylation site 34.50% 50.22% 11.79% 3.49% 229 7.18
PS00005 Protein kinase C phosphorylation site 31.95% 46.75% 21.30% 0.00% 169 4.79
PS00001 N-glycosylation site 31.13% 39.62% 26.42% 2.83% 106 5.18
PS00007 cAMP- and cGMP-dependent protein kinase phosphorylation site 42.11% 42.11% 5.26% 10.53% 19 2.46
PS00009 Amidation site 15.79% 68.42% 15.79% 0.00% 19 1.25
PS00430 TonB-dependent receptor proteins signature 100.00% 0.00% 0.00% 0.00% 4 8.59
PS00013 100.00% 0.00% 0.00% 0.00% 4 8.59Prokaryotic membrane lipoprotein lipid attachment site
Beta-barrel protein PROSITE motif assignments
my $im = DrawTransmembrane->new(-title=>'CLN3 topology prediction using MEMSAT3_6', -n_terminal=>'in',
-topology=>'37,56,100,119,129,153,200,224,280,299,353,375', -labels=> \%labels, -outside_label=>'Lumen', -inside_label=>'Cytoplasm', -membrane_label=>'Membrane');
print OUTPUT $im->png;
A Bioperl module to draw transmembrane proteins
Conclusions
I have successfully achieved my major goal for the first 6 months - to create a high quality dataset of transmembrane proteins of known topology.
My second goal - to scan the novel data set against motif and domain databases to identify signatures which were consistently located on either inside or outside loops - has also been completed.
In collaboration with Dr Sara Mole (MRC Laboratory for Molecular Cell Biology), I have begun an analysis of CLN3 (Batten's Disease protein) with a view to predicting the protein's topology using a combination of computational and experimental evidence.
I have written a module using Perl to create graphical representations of transmembrane proteins given the positions of their transmembrane helices and the location of the N-terminal. This module has been accepted by the Bioperl project and will be available in the next release.