Multiple alignment

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Multiple alignment
Peter Højrup Department of Biochemistry
Molecular Biology, SDU, Odense University,
Denmark.
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Multiple sequence alignment
One amino acid sequence plays coy A pair of
homologous sequences whisper Many aligned
sequences shout out loud.
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Main applications for MA

• Extrapolation
• Determine family relationship
• Phylogenetic analysis
• Reconstruct the history of the protein
• Pattern identification
• Identify structural/functional important residues
• Domain identification
• Construct a pattern to find other family members
• Structure prediction
• A multiple alignment greatly enhances prediction
  accuracy
• PCR analysis
• Find the less degenerate parts of a gene.

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Definition
Consensus sequence
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Multiple alignment parameters

• AA substitution matrix
• Usually one based on global alignment PAM250 or
  Gonnet
• Gap parameters
• Both opening and extension parameters are very
  important for the optimal alignment
• Alignment order
• ClustalX initially performs a pairwise comparison
  closest fit is the initial alignment.
• Sequence length
• Try to align sequences of the same length you
  may do an initial dot-plot for alignment regions.

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Multiple sequence alignment
Rat_CALRETICULIN ----MLLSVPLLLGLLGLAAAD-------
---------------------------PAIYFKEQFLDGDAWTNR-----
----WVESKHKSD--FGKFVL Human_CALRETICULIN
----MLLSVPLLLGLLGLAVAE----------------------------
------PAVYFKEQFLDGDGWTSR---------WIESKHKSD--FGKFVL
RAT_CALNEXIN MEGKWLLCLLLVLGTAAIQAHDGHDDD
MIDIEDDLDDVIEEVEDSKSKSDTSTPPSPKVTYKAPVPTGEVYFADSFD
RGSLSGWILSKAKKDDTDDEIAK Human_CALNEXIN
MEGKWLLCMLLVLGTAIVEAHDGHDDDVIDIEDDLDDVIEEVEDSKPDT-
TAPPSSPKVTYKAPVPTGEVYFADSFDRGTLSGWILSKAKKDDTDDEIAK
. .
.
. .. Prim.cons.
MEGK2LL2V2L2LG22GLAA2DGHDDD2IDIEDDLDDVIEEVEDSK222D
T22P2SP2V22K22222G2V22A2SFDRG2LSGWI2SK2K2DDT222222
Rat_CALRETICULIN SSGKFYGDQEK------DKGLQTSQD
ARFYALSARF-EPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPGG--LD
QKDMHGDSEYNIMFGPDICGPGTK Human_CALRETICULIN
SSGKFYGDEEK------DKGLQTSQDARFYALSASF-EPFSNKGQTLVVQ
FTVKHEQNIDCGGGYVKLFPNS--LDQTDMHGDSEYNIMFGPDICGPGTK
RAT_CALNEXIN YDGKWEVDEMKETKLPGDKGLVLMSRA
KHHAISAKLNKPFLFDTKPLIVQYEVNFQNGIECGGAYVKLLSKTSELNL
DQFHDKTPYTIMFGPDKCG-EDY Human_CALNEXIN
YDGKWEVEEMKESKLPGDKGLVLMSRAKHHAISAKLNKPFLFDTKPLIVQ
YEVNFQNGIECGGAYVKLLSKTPELNLDQFHDKTPYTIMFGPDKCG-EDY
. .
. . . ....
.. . Prim.cons.
22GK222DE2KE2KLPGDKGL22222A222A2SAK2N2PF222222L2VQ
22V22222I2CGG2YVKL22KT2EL22D22H2222Y2IMFGPD2CGP222
Rat_CALRETICULIN KVHVIFNYKGKNVLINKDIRCK----
------DDEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWD--FLPPK
KIKDPDAAKPEDWDERAKIDDPTD Human_CALRETICULIN
KVHVIFNYKGKNVLINKDIRCK----------DDEFTHLYTLIVRPDNTY
EVKIDNSQVESGSLEDDWD--FLPPKKIKDPDASKPEDWDERAKIDDPTD
RAT_CALNEXIN KLHFIFRHKNPKTGVYEEKHAKRPDAD
LKTYFTDKKTHLYTLILNPDNSFEILVDQSVVNSGNLLNDMTPPVNPSRE
IEDPEDRKPEDWDERPKIADPDA Human_CALNEXIN
KLHFIFRHKNPKTGIYEEKHAKRPDADLKTYFTDKKTHLYTLILNPDNSF
EILVDQSVVNSGNLLNDMTPPVNPSREIEDPEDRKPEDWDERPKIPDPEA
... . .
. . .
. . Prim.cons.
K2H2IF22K22222I222222KRPDADLKTYF2D22THLYTLI22PDN22
E222D2S2V2SG2L22D22PP22P222I2DP22RKPEDWDER2KIDDPT2
Rat_CALRETICULIN SKPEDWDK------------------
---PEHIPDPDAKKPEDWDEEMDGEWEP-------------------PVI
QNPEYKGEWKPRQIDNPDYKGTWI Human_CALRETICULIN
SKPEDWDK---------------------PEHIPDPDAKKPEDWDEEMDG
EWEP-------------------PVIQNPEYKGEWKPRQIDNPDYKGTWI
RAT_CALNEXIN VKPDDWDEDAPSKIPDEEATKPEGWLDD
EPEYIPDPDAEKPEDWDEDMDGEWEAPQIANPKCESAPGCGVWQRPMIDN
PNYKGKWKPPMIDNPNYQGIWK Human_CALNEXIN
VKPDDWDEDAPAKIPDEEATKPEGWLDDEPEYVPDPDAEKPEDWDEDMDG
EWEAPQIANPRCESAPGCGVWQRPVIDNPNYKGKWKPPMIDNPSYQGIWK


.
. Prim.cons.
2KP2DWD2DAP2KIPDEEATKPEGWLDDEPE2IPDPDA2KPEDWDE2MDG
EWE2PQIANP2CESAPGCGVWQRPVI2NP2YKG2WKP22IDNPDY2G2W2

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Nomenclature
Rat_CALRETICULIN SSGKFYGDQEK------DKGLQTSQDARF
YALSARF-EPFSNKGQTL Human_CALRETICULIN
SSGKFYGDEEK------DKGLQTSQDARFYALSASF-EPFSNKGQTL RA
T_CALNEXIN YDGKWEVDEMKETKLPGDKGLVLMSRAKHHA
ISAKLNKPFLFDTKPL Human_CALNEXIN
YDGKWEVEEMKESKLPGDKGLVLMSRAKHHAISAKLNKPFLFDTKPL
. .
. . . Prim.cons.
22GK222DE2KE2KLPGDKGL22222A222A2SAK2N2PF222222L
Consensus sequence
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Mind the gap!
250 260 270
280 290 300

Papain DGVRQVQPYNEGALLYSIANQPVSVVLEAAGKDFQ
LYRGGIFVGPCGNKVDHAVAAVGYG Staphopain
--------I---AILGSRV-E-----S----------RNGMHAGHAMAVV
GN--AKLNNG .
... . . . Prim.cons.
DGVRQVQP2NEGA2L2S22N2PVSVV2EAAGKDFQLYR2G222G22222V
22AVA2222G
Never have islands (widows)
Gaps should be in-frame
Papain YTTTELSYEEVLNDGDVNIPEYVDWRQKGAVTPVKNQ
GSCGSCWAFSAVVTIEGIIKIRT CathLx2
PRKGKVFQEPLFYEA----PRSVDWREKGYVTPVKNQGQCGSCWAFSATG
ALEGQMFRKT CathBx3 PPQRVMFTEDLKLPAS--FDAREQWP
QCPTIKEIRDQGSCGSCWAFGAVEAISDRICIHT Staphopain
---------------------ETQGNN-------------GWCAGYTMSA
LLN-------
. . Prim.cons.
P33333F3E3L333A2VN2P34V2WRQKG3VTPVKNQGSCGSCWAFSAV4
A2EG3I3I3T
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Structural inferences

• The most highly conserved regions are likely to
  correspond to the active site.
• Regions rich in insertions and deletions probably
  correspond to surface loops.
• A position containing a conserved Gly or Pro
  probably corresponds to a turn.
• A conserved pattern of hydrophobicity with
  spacing 2 (that is every second residue) with
  the intervening residues more variable and
  including hydrophilic residues suggests a
  b-strand on the surface.
• A conserved pattern of hydrophobicity with
  spacing 4 suggests a (surface) a-helix.

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ClustalW/ClustalX

• Multiple alignment takes place in three steps
• Pairwise alignment of all sequences
• Calculating a guide tree
• Progressive alignment.

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Guide tree of hexokinases
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Hexokinase alignment
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3D aspects - thioredoxins
Loop
a
Loop
Loop
Active site
Loop
b
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Thioredoxin
Loop
Loop
a
b
Loop
Loop
Active site
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Sequence logo from alignment
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Naming conventions in multiple alignments

• Clustal W only use the first word (i.e. never use
  white space in name)
• Do not use special symbols use underscore _
  to connect words
• The protein should be understandable in 15
  characters (truncation)
• All proteins to be aligned needs an individual
  name.

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PSI - BLAST

• Position Iterated Blast
• For each search round, the aligned results are
  used as the basis for calculating a new
  substitution matrix.
• New iterations can be carried out as long as new
  hits are found.
• If no results are found in a normal BLAST,
  PSI-BLAST will not help.
• Check results carefully!!

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PSI BLAST of HIT protein
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First search 103 hits
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First PSI iteration
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Second PSI iteration