Corrections
SEQUENCE 4
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>seq4
MSTNNYQTLSQNKADRMGPGGSRRPRNSQHATASTPSASSCKEQQKDVEH
EFDIIAYKTTFWRTFFFYALSFGTCGIFRLFLHWFPKRLIQFRGKRCSVE
NADLVLVVDNHNRYDICNVYYRNKSGTDHTVVANTDGNLAELDELRWFKY
RKLQYTWIDGEWSTPSRAYSHVTPENLASSAPTTGLKADDVALRRTYFGP
NVMPVKLSPFYELVYKEVLSPFYIFQAISVTVWYIDDYVWYAALIIVMSL
YSVIMTLRQTRSQQRRLQSMVVEHDEVQVIRENGRVLTLDSSEIVPGDVL
VIPPQGCMMYCDAVLLNGTCIVNESMLTGESIPITKSAISDDGHEKIFSI
DKHGKNIIFNGTKVLQTKYYKGQNVKALVIRTAYSTTKGQLIRAIMYPKP
ADFKFFRELMKFIGVLAIVAFFGFMYTSFILFYRGSSIGKIIIRALDLVT
IVVPPALPAVMGIGIFYAQRRLRQKSIYCISPTTINTCGAIDVVCFDKTG
TLTEDGLDFYALRVVNDAKIGDNIVQIAANDSCQNVVRAIATCHTLSKIN
NELHGDPLDVIMFEQTGYSLEEDDSESHESIESIQPILIRPPKDSSLPDC
QIVKQFTFSSGLQRQSVIVTEEDSMKAYCKGSPEMIMSLCRPETVPENFH
DIVEEYSQHGYRLIAVAEKELVVGSEVQKTPRQSIECDLTLIGLVALENR
LKPVTTEVIQKLNEANIRSVMVTGDNLLTALSVARECGIIVPNKSAYLIE
HENGVVDRRGRTVLTIREKEDHHTERQPKIVDLTKMTNKDCQFAISGSTF
SVVTHEYPDLLDQLVLVCNVFARMAPEQKQLLVEHLQDVGQTVAMCGDGA
NDCAALKAAHAGISLSEAEASIAAPFTSKVADIRCVITLISEGRAALVTS
YSAFLCMAGYSLTQFISILLLYWIATSYSQMQFLFIDIAIVTNLAFLSSK
TRAHKELASTPPPTSILSTASMVSLFGQLAIGGMAQVAVFCLITMQSWFI
PFMPTHHDNDEDRKSLQGTAIFYVSLFHYIVLYFVFAAGPPYRASIASNK
AFLISMIGVTVTCIAIVVFYVTPIQYFLGCLQMPQEFRFIILAVATVTAV
ISIIYDRCVDWISERLREKIRQRRKGA
Compute pI/Mw tool
!!! If you choose the wrong format for the sequence…
With the correct format:
ProtParam
• SAPS
SAPS (1)
SAPS (2)
doi: 10.1093/bioinformatics/bti797
http://en.wikipedia.org/wiki/Coiled_coil
The coiled-coil domains are
annotated according to 3D
structure data (experimental
data)
Coiled-coil prediction
• Coils
Coils prediction
http://www.ch.embnet.org/software/COILS_form.html
Coiled-coil prediction
• PairCoil (not always working…)
Paircoil prediction
Coiled-coil prediction
• PairCoil2
Parcoil2 results
Coiled-coil prediction
• Sliding window (Protscale)
Sliding window
amino acid scaleexample:
• Bad results----
Bad results….
Sliding windows and amino acid scales
Transmembrane domain: alpha-helix of 20
amino acids (hydrophobic)
• -> amino acid scales: hydrophobicity and
alpha helix
• -> sliding window size: 20 amino acids
Protscale
Amino acid scale: Kyte and Doolittle (hydrophobicity)
Sliding window size: 21 amino acids
Protscale
Amino acid scale: Chou&Fasman (alpha helix)
Sliding window size: 21
Sliding windows and amino acid scales
Transmembrane domain: alpha-helix of 20
amino acids (hydrophobic)
• -> amino acid scales: hydrophobicity and
alpha helix
• -> sliding window size: 20 amino acids
Method based HMM or NN
HMMTOP
HMMTOP
• Protein: seq4
• Length: 1127
• N-terminus: IN
• Number of transmembrane helices: 8
Transmembrane helices: 65-82 409-432
445-468 916-940 970-993 1020-1039 10521072 1089-1106
TMHMM (1)
TMHMM (2)
TMpred (1)
PSORT II (1)
- Look for the presence of a signal peptide.
No signal peptide
Signal peptides are
often predicted as
‘transmembrane’
domains (or vice versa)
as they amino acids
with similar
biochemical properties
(hydrophic and alpha
helix).
Transmembrane: resume
HMMTOP (8 TM)
1
in
Big loop
1130
PSORT II (10 TM)
1
1130
Tmpred (10 TM)
1
in
1130
TMHMM (11 TM)
1 out
1130
? missed TM
P39986
O14072
Q9HD20
Q9Y139
P90747
Q9LT02
Q12697
O74431
Q9N323
Q27533
Q21286
Q9NQ11
O14022
Q95050
Q9XXW1
Consensus
Segment
Helix
...........mtkksfvsspivrdstllvpksliakpyvlpffplyatfaqlyfqqydryikgpewtfvylgtlvslnilvmLmpaW.nvkikakfny.........
...........mgskalitspdissgqlyiklptffhlyvwpfalfvypyigyvyqnklyseevryltyiavg...tihalfwLageW.ntkvyclmtc.........
..........................................................................................mekweelnshqp......
lvqyvslhvriptpltgvvlpfvplylsafylwinvtggqendttnndvitadnqtttdnittwndvgfigvlaiaflhiltlLfcyW.svhvlafltc.........
................mgvdqlvetiipynlrsiathlyvppftiitaiwtyvwlnifgyeey.yelgmlgyaaifvilalvlLfchW.mmpvrcflmc.........
............mssfrvggkvvekvdlcrkkqlvwrldvwpfailytvwlttivpsidfsd.....acialgglsafhilvlLfttW.svdfkcfvqfskmnlellv
rsasennrgsfsghddvhnqhseylkpdyhekfypqyapnlhyqrfyiaeedlvigiaayqts.kfwyiiynlccfltfglvyLltrW.lphlkvkly..........
ngsgvysdeeeitemmleelnihpvlrresvgeaaglsedgccqilylveedlevgiagyktn.ksryrlyqaiclltlglayLifrW.lpkyfirfv..........
...mstnnyqtlsqnkadrmgpggsrrprnsqhatastpsassckeqqkdvehefdiiayktt.fwrtfffyalsfgtcgifrLflhW.fpkrliqfr..........
.............................................mtlesgdhtltlfayrtg.pfrtilfyaltvltlgifrLilhW.kqkwdvkmr..........
mtsereplldtttrnrvydttdnpstkimkrekdnpkakttsfnqgklnigeetcdlyayket.igrqilfwlltivtlgfyqLlayW.vkslfvkvr..........
....................msadssplvgstptgygtltigtsidplsssvssvrlsgycgs.pwrvigyhvvvwmmagiplLlfrW.kplwgvrlr..........
...................mdsielkqlvpendsepgtprqllfqhydisneetigikpfksi.pakvyilrvteiltlgllhLiltW.lpefrlkwi..........
...........................mrvssieaemenpidvdktdvegelkikqvtllren.........ivkkivfflvaifcsd.rpsvlkkvfy.........
aleffiffllsltitygiliirkhiqslflkpsllkdsdyviiytineeyntfyntnyfkkyishinhmihtfikkkkknikknikkWnfqkynilflqfvcnlldii
-----------------------------------------------------------------------------------L---W--------------------------------------------------------------------------------------------------------------------------------------------------------------aaaaaaaaaaaaaaaaaaaaaa-------------bbbbbbbbbbbbbbbbbbbbb--------------------
• The protein is known to contain 12 TM: one TM is missing at the N-terminus
• The possible ways to find the correct protein topology is to do a multiple alignment
with other family members, or to do some 3D experiment (which are difficult with
proteins containing transmembrane domains)
SEQ4 = Q9N323
Kristian Axelsen: personnal communication
P0AER0
The Aquaglyceroporin contains ½ transmembrane regions which
can not be predicted by programs, because the region is too short
(less than 20 amino acids). There is no way to predict such
transmembrane regions, except by doing 3D experiments. 3D
experiments is the only way to confirm and ‘predict’ correctly
transmembrane domains. Similarity analysis could then help to
predict such regions in other protein of the same family.
M3 and M7 are ‘demi’ transmembrane: not predictable
http://www.uniprot.org/uniprot/P0AER0#section_features
Look for the transmembrane regions of P31243 (try the
different transmembrane prediction programs): your
conclusions ?
No transmembrane domains are found by any program
because this protein, a porin, is anchored in the membrane
by a specific 3D structure called beta barrel which does not
have any alpha helix….
‘beta barrel’
Mainly composed of beta-sheets in a 16-stranded beta-barrel formation and forms a
pore in the membrane 1.7 - 2.5 nm in diameter. Note that the orientation of the strands
is such that side chains alternately point into the interior and exterior of the pore; the
former are strongly polar residues while the latter are very hydrophobic.
Beta barrel
Porin from Rhodobacter
Alignment of the 2 isoforms
The gene has two in-frame initiation codons and two
different proteins are made by alternative initiation (of
translation)
According to this publication (PubMed:
11274159), there is a 'Dual targeting of spinach
protoporphyrinogen oxidase II to mitochondria
and chloroplasts by alternative use of two inframe initiation codons'.
Immunoblot analysis of Protox II in spinach leaf.
Total leaf
Watanabe N et al. J. Biol. Chem. 2001;276:20474-20481
©2001 by American Society for Biochemistry and Molecular Biology
chloro
mito
Q94IG7 – Long isoform
• wolfPSORT: chloroplast
• TargetP: chloroplast
• CH score: 0.826
• MI score: 0.026
• ER score: 0.101
• Other location: 0.060
• SignalP-NN: not secreted
• score (D): 0.285
• SignalP-HMM: not secreted
• SP probability: 6.2%
• SA probability: 0.2%
• ChloroP: chloroplast
• prediction score: 0.549
• MITOPROT: mitochondria !!!
exported to mitochondria with
a probability of 0.71 !!!!
Q94IG7 – Short isoform
• wolfPSORT: mitochondrial
• TargetP: mitochondrial
• CH score: 0.123
• MI score: 0.504
• ER score: 0.048
• Other location: 0.400
• SignalP-NN: not secreted
• score (D): 0.195
• SignalP-HMM: not secreted
• SP probability: 3.1%
• SA probability: 5%
• ChloroP: not in chloroplast
• prediction score: 0.473
• MITOPROT: other location
• exported to mitochondria with a
probability of 0.33 !!!!!!
Cystein (61 modifications) and serine (46 modifications)
are the amino acids with the highest number of known associated
PTM.
Beware: Resid considers the selenocystein as a PTM…this is
not the case !
Phosphorylation
P03372
P03372
P03372
UniProt data: Experimentally proved
http://www.phosphosite.org/proteinAction.do?id=968&showAllSites=true
The phosphorylation sites are localized on the ‘surface’ of the protein
(homodimer) (where the amino acid are accessible to the kinases !)
O-glycosylation
P02724
Myristoylation
P51876
P51876
NMT
Myristoylator
(Not predictable…)
Protein: secreted protein
(P02751, fibronectin)
(predictable…)
(predictable…)
(Not predictable…)
Can be predicted:
-Subcellular location (PSORT, TargetP)
-Domains (InterPro)
-Signal
-Sulfation
-N-glycosylation
-O-glycosylation
-Phosphorylation
(Not predictable…)
THE END