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Exploring Protein Sequences – Part 1
Part 1:
Part 2:
Patterns and Motifs
Profiles
Hydropathy Plots
Transmembrane helices
Antigenic Prediction
Signal Peptides
Repeats
Coiled Coils
Protein Domains
Domain databases
Celia van Gelder
CMBI
Radboud University
December 2005
©CMBI 2005
Patterns and Motifs (1)
•In a multiple sequence alignment (MSA) islands of conservation
emerge
•These conserved regions (motifs, segments, blocks, features) are
typically around 10-20 aa in length
•They tend to correspond to the core structural or functional
elements of the protein
•Their conserved nature allows them to be used to diagnose family
membership
©CMBI 2005
Patterns and Motifs (2)
•A motif (or pattern or signature) is a regular expression for what
residues can be present at any given position.
•Motifs can contain
- alternative residues
- flexible regions
C-x(2,5)-C-x-[GP]-x-P-x(2,5)-C
CXXXCXGXPXXXXXC
|
| | |
|
FGCAKLCAGFPLRRLPCFYG
Syntax:
A-[BC]-X-D(2,5)-{EFG}-H
Means:
A
B or C
Anything
2-5 D’s
Not E,F or G
H
Patterns and Motifs (3)
•Motifs can not contain
- mismatches
exact match or no match at all
- gaps
C-x(2,5)-C-x-[GP]-x-P-x(2,5)-C
CXXCXGXPXXXXX-C
| ?| | |
?|
FGCA-CAGFPLRRLPKCFYG
J.Leunissen
PROSITE
• PROSITE - A Dictionary of Protein Sites and Patterns
• 1328 patterns and 577 profiles/matrices (dec 2005)
• For every pattern or profile there is documentation present (e.g.
PDOC00975)
- information on taxonomic occurrence
- domain architecture,
- function,
- 3D structure,
- main characteristics of the sequence
- some references.
©CMBI 2005
PROSITE Pattern
•PROSITE patterns consist of an exact regular expression
•Possible patterns occur frequently in proteins; they may not
actually be present, such as post-translational modification sites
ID ASN_GLYCOSYLATION; PATTERN.
DE N-glycosylation site.
PA N-{P}-[ST]-{P}.
•Notice also in the PROSITE record the number of false positives
and false negatives
©CMBI 2005
PROSITE Pattern (2)
©CMBI 2005
Profiles
•If regular expressions fail to define the motif properly we need a
profile.
•Profiles are specific representations that incorporate the entire
information of a multiple sequence alignment.
•A profile is a position-specific scoring scheme and holds for each
position in the sequence 20 scores for the 20 residue types, and
sometimes also two values for gap open and gap elongation.
•Profiles provide a sensitive means of detecting distant sequence
relationships
©CMBI 2005
©CMBI 2005
Hydropathy plots
Hydropathy plots are designed to display the distribution of polar and
apolar residues along a protein sequence.
A positive value indicates local hydrophobicity and a negative value
suggests a water-exposed region on the face of a protein.
(Kyte-Doolittle scale)
Hydropathy plots are generally most useful in predicting transmembrane
segments, and N-terminal secretion signal sequences.
©CMBI 2005
Hydropathy scales
©CMBI 2005
Sliding Window Approach
Sum amino acid property (e.g. hydrophobicity values) in a given
window
Plot the value in the middle of the window
I L I K E I R
4.50+3.80+4.50-3.90-3.50+4.50-4.50 = 5.40
=>
5.4/7=0.77
Move to the next position in the sequence
L I K E I R Q
+3.80+4.50-3.90-3.50+4.50-4.50 – 3.50 =
=>
-2.6/7=-0.37
J. Leunissen
Hydropathy plot
for rhodopsin
The window size can be changed. A small window produces "noisier" plots that more
accurately reflect highly local hydrophobicity.
A window of about 19 is generally optimal for recognizing the long hydrophobic
stretches that typify transmembrane stretches.
©CMBI 2005
Transmembrane Helices
Transmembrane proteins are integral membrane proteins that interact
extensively with the membrane lipids.
Nearly all known integral membrane proteins span the lipid bilayer
Hydropathy analysis can be used to locate possible transmembrane
segments
The main signal is a stretch of hydrophobic and helix-loving amino acids
©CMBI 2005
Transmembrane Helices (2)
In a -helix the rotation is 100 degrees per amino acid
The rise per amino acid is 1,5 Å
To span a membrane of 30 Å approx. 30/1,5 = 20 amino acids are
needed
©CMBI 2005
Transmembrane Helix Prediction Servers
1. KDD
2. Tmpred (database Tmbase)
3. DAS
4. TopPred II
5. TMHMM 2.0
6. MEMSAT 2
7. SOSUI
8. HMMTOP 2.0
©CMBI 2005
Antigenic Prediction
General Remarks
Antibodies are a powerful tool for life science research
They find multiple application in a variety of areas including biotechnology,
medicine and diagnosis.
Antibodies can recognize either linear or 3D epitopes
There are rules to predict what peptide fragments from a protein are likely
to be antigenic
©CMBI 2005
Antigenic Prediction
1. Antigenic peptides should be located in solvent accessible
regions and contain both hydrophobic and hydrophilic residues
•
•
Determine solvent accessibility in case 3D coordinates are
available.
If you have only a sequence, predict the accessibilities.
2. The peptide should also adopt a conformation that mimics its
shape when contained within the protein.
•
•
Preferably select peptides lying in long loops connecting
secondary structure motifs.
Neither the peptide stand-alone, nor the peptide in the full protein
should be helical.
©CMBI 2005
Rules of thumb in antigenic prediction
•N- and C- terminal peptides sometimes work better than peptides
elsewhere in the protein.
•Avoid peptides with internal sequence repeats or near repeats.
•Avoid sequences that look funny (i.e. avoid low complexity sequences).
•Try to avoid prolines and cysteines.
•Last, but not least, use antigenicity prediction programs.
©CMBI 2005
Signal Peptides
Proteins have intrinsic signals that
govern their transport and
localization in the cell (nucleus, ER,
mitochondria, chloroplasts)
Specific amino acid sequences
determine whether a protein will
pass through a membrane into a
particular organelle, become
integrated into the membrane, or
be exported out of the cell.
©CMBI 2005
Signal Peptides (2)
The common structure of signal peptides from various proteins is
described as:
• a positively charged (N-terminal) n-region
• followed by a hydrophobic h-region (which can adopt an -helical
conformation in an hydrophobic environment)
• and a neutral but polar c-region (cleavage region; the signal
sequence is cleaved off here after delivering the protein at the
right site).
The (-3, -1) rule states that the residues at positions –3 and –1 (relative to
the cleavage site) must be small and neutral for cleavage to occur
correctly.
©CMBI 2005
Signal Peptides (3)
Eukaryotes
Total length
(average)
n-regions
h-regions
c-regions
-3,-1 positions
+1 to +5 region
22.6 aa
only slightly Arg-rich
short, very
hydrophobic
short, no pattern
small and neutral
residues
no pattern
Prokaryotes
Gram-negative
Gram-positive
25.1 aa
32.0 aa
Lys+Arg-rich
slightly longer, less
very long, less
hydrophobic
hydrophobic
short, Ser+Ala-rich
longer, Pro+Thr-rich
almost exclusively Ala
rich in Ala, Asp/Glu, and Ser/Thr
Marlinda.Hupkes 2004
Prediction of Signal Peptides
Prokaryotes and Eukaryotes:
SignalP 3.0
SPScan
SigCleave
PSORT
Eukaryotes:
SIGFIND
TargetP
Specific localization signals:
PredictNLS - Nuclear Localization Signals
ChloroP – Chloroplast transit peptides
NetNes – Nuclear Export Signals
©CMBI 2005
Repeats in proteins
•Although they are usually found in non-coding genomic regions, repeating
sequences are also found within genes.
•Ranging from repeats of a single amino acid, through three residue short
tandem repeats (e.g. in collagen), to the repetition of homologous domains
of 100 or more residues.
•Duplicated sequence segments occur in 14 % of all proteins, but
eukaryotic proteins are three times more likely to have internal repeats
than prokaryotic proteins
©CMBI 2005
Repeats, example 1
Ewan Birney
Repeats, example 2
©CMBI 2005
Prediction of Repeats
•
Repsim (a database of simple repeats)
•
Rep (Searches a protein sequence for repeats)
•
RADAR (Rapid Automatic Detection and Alignment of Repeats in
protein sequences.)
•
REPRO (De novo repeat detection in protein sequences)
•
Other?
©CMBI 2005
Coiled-Coils
The coiled-coil is a ubiquitous protein motif that is often used to control
oligomerisation.
It is found in many types of proteins, including transcription factors, viral
fusion peptides, and certain tRNA synthetases.
Most coiled-coil sequences contain heptad repeats - seven residue
patterns denoted abcdefg in which the a and d residues (core positions)
are generally hydrophobic.
A number of programs are available to predict coiled-coil regions in a
protein: COILS, PAIRCOILS, MULTICOILS.
©CMBI 2005