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Motif
The term motif is used in two different ways in structural biology.
First
 Refers to a particular amino-acid sequence that is characteristic of a
specific biochemical function.
 For example, Zinc finger motif CXXCXXXXXXXXXXXXHXXXH,
which is found in a widely varying family of DNA-binding proteins.
 The conserved cysteine and histidine residues in this sequence motif form
ligands to a zinc ion, which is essential to stabilize the tertiary structure.
 Conservation is sometimes of a class of residues rather than a specific
residue: for example, in the 12-residue loop between the zinc ligands, one
position is preferentially hydrophobic, specifically leucine or phenylalanine.
 Sequence motifs can often be recognized by simple inspection of the
amino-acid sequence of a protein, and when detected provide strong evidence
for biochemical function. For example, the protease from the human
immunodeficiency virus was first identified as an aspartyl protease because a
characteristic sequence motif for such proteases was recognized in its
primary structure.
Motif
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Second
 Motif can refer to a set of contiguous secondary structure elements that either
have a particular functional significance or define a portion of an independently
folded domain.
 The elements with the functional sequence motifs are known as functional
motifs. An example is the helix-turn-helix motif found in many DNA-binding
proteins.
 This simple structural motif will not exist as a stably folded domain if
expressed separately from the rest of its protein context, but when it can be
detected in a protein that is already thought to bind nucleic acids, it is a likely
candidate for the recognition element. Examples -The Rossmann fold, an alpha/beta twist arrangement that usually binds NAD
cofactors;
- The Greek-key motif, an all-beta-sheet arrangement found in many different
proteins and which topologically resembles the design found on ancient vases.
 As these examples indicate, these structural motifs sometimes are suggestive
of function, but more often are not: the only case here with clear functional
implications is the Rossmann fold.
Identification of functional peptide in a protein
(http://motif.genome.jp/)
Continued..
Output of a motif search of IL-22
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Pairwise alignment
 The most important question about a gene or protein is whether it is related
to any other gene or protein.
 Relatedness of two proteins at the sequence level suggests that they are
homologous and may have common functions.
Pairwise alignment is the process of lining up two sequences to achieve
maximal levels of identity.
 By analyzing many DNA and protein sequences, it is possible to identify
domains or motifs that are shared among a group of molecules.
 These analyses of the relatedness of proteins and genes are accomplished
by aligning sequences
 The complete genome sequences of many organisms’ and their alignments
help us to find how proteins are related within an organism and between
organisms. These becomes fundamental to our understanding of life.
 Finally, whether two proteins are homologous comes from structural
studies in combination with evolutionary analyses.
Protein Alignments are more informative than DNA alignments
 Between the choice of aligning a DNA sequence or the sequence of the protein
it encodes, comparison of protein sequence is usually more informative. There
are several reasons for this –
1. Many changes in a DNA sequence particularly at the third position of a codon
do not change the amino acid that it specified
2. Many amino acid share related biophysical properties e.g., lysine and arginine
are both basic amino acids. The important relationships between related but
mismatched amino acids in an alignment can be accounted for using scoring
systems.
3. Protein sequence comparisons can identify homologous sequences from
organisms that last shared a common ancestor over 1 billion years ago e.g.,
glutathione transferase. In contrast, DNA sequence comparisons typically
allow lookback times of up to about 600 million years ago.
4. We can easily move between the worlds of DNA and protein by the tblastn tool
from the NCBI BLAST website
 Nevertheless, in many cases it is appropriate to compare nucleotide sequences
1. In searching for polymorphisms
2. In analyzing the identity of a cloned cDNA fragment
Assesment of two proteins by pairwise alignment
 It is possible to asses the relatedness of two proteins by performing a
pairwise alignment by placing two sequences directly next to each other.
 It is extremely difficult to align these two proteins by visual inspection.
 If we allow gaps in the alignment to account for deletions or insertions
in the two sequences, the number of possible alignments rises
exponentially.
 An algorithm can help us to perform an alignment (e.g., heuristic
algorithm). GAP program of the genetics computer group (GCG). This
program use heuristic algorithm to do this.
Pairwise alignment of human RBP and bovine -lactoglobulin
 Along the top row the residues GTWY are all identical between the two
proteins. The program also count the number of identical residues, in this
case the protein share 26% identity (43 residues).
  – identical, 1) paired dots – replacement with similar residues but not
identical because they share similar biological properties, Arg (R), Lys (K);
2) Single dots between aligned residues also indicate similarity but less than
for paired dots. 3,4) gaps 5) dot above the sequences indicate every 10 bp.
Percent identity and percent similarity
 The percent similarity of two protein sequences is the sum of both
identical and similar matches.
 In the alignment shown before there are 44 aligned amino acid residues
of which 11 are identical and 3 are similar. The percent identity is 25%
(11/14) and the percent similarity is 32% (14/44).
 In general, it is more useful to consider the identity shared by two
protein sequences, rather than the similarity, because the similarity measure
may be based upon a variety of definitions of how similar two amino acids
residues are to each other.
Pairwise Alignment, Homology and Evolution of life
 If two proteins are homologous, they share a common ancestor.
 Generally, we observe the sequence of proteins from organisms that are
extant.
 We can compare RBP from species such as human or fish, rainbow
trout, and see that the sequences are homologous.
 This implies that an ancestral organism had an RBP gene and lived
sometime before the divergences of the lineages that gave rise to human
and trout.
 The study of homologous
protein sequences by
pairwise alignment involves
an investigation of the
evolutionary history of that
protein.
 For the brief overview of the time scale of life on earth we see that
the divergence of different species is established through the use of
many sources of data, especially the fossil record.
 Consider the time scale of life
on earth.
 Fossils of procaryotes have
been discovered in rocks 3.5
billion years old.
 In the case of lipocalins, no
invertebrate (e.g., insect)
ortholog of RBP has been
identified, but several fish and
amphibian RBPs are known.
 So, it can be inferred that the
RBP gene originated between
700 and 400 MYA.
Other lipocalins are more
ancient like bacterial lipocalin
genes presumably arose 2 BYA.
 Homologous protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
enzyme is well conserved through the evolution and very ancient.
 Orthologous RBPs from another species provide another example of well
conserved family.
 Many columns in this alignment are perfectly conserved, including the
glycine-X-tryptophan (GXW) motif that is characteristics of lipocalin proteins.
 Some positions are less well conserved like immediately preceding the
canonical GXW motif, the amino acid may be glutamine, threonine, serine, or
alanine.
 Amino acid residues that form a binding pocket for retinol are perfectly
conserved (showed in arrow).
 Despite the tremendous divergence of the amino acid sequences, it is likely
that all members of this family adopt a highly similar three-dimensional
structure.
DAYHOFF MODEL: ACCEPTED POINT MUTATIONS
 Dayhoff and colleagues catalogued thousands of proteins and compared
the sequences of closely related proteins in many families.
 They considered the question of which specific amino acid substitutions
are observed to occur when two homologous protein sequences are
aligned.
 They defined an accepted point mutation (PAM) as a replacement of one
amino acid in a protein by another residue that has been accepted by
natural selection.
 An amino acid change that is accepted by natural selection occurs wheni) a gene undergoes DNA mutation such that it encodes a different amino
acid
ii) the entire species adopts that change as the predominant form of the
protein
Specific point mutations accepted in protein in evolution:
 Dayhoff and colleagues examined 1572 changes in 71 groups of closely
related proteins.
 Thus their definition of accepted mutations was based on empirically
observed amino acid changes.
 Conservative replacement such as serine for threonine are most readily
accepted during evolution
 Dayhoff et al., calculated the relative mutabilities of the amino acids.
 This simply describes how often each amino acid is likely to change over
a short evolutionary period.
 Gonnet and others have produced updates versions of PAM matrices.
 They found similar data to that of Dayhoff. Some amino acid residues
such as asparagine and serine undergo substitution very frequently while
tryptophan and cysteine are mutable only very rarely.
Why are some amino acids more mutable than others?
 The less mutable residues probably have important structural
and functional roles in proteins, such that the consequence of
replacing them with any other residue could be harmful to the
organism.
 Conversely, the most mutable amino acids – asparagine, serine,
aspartic acid, glutamic acid – have functions in proteins that are
easily assumed by other residues. The most common
substitutions are –
glutamic acid for aspartic acid (both are acidic),
serine for alanine,
serine for threonine (both are hydroxylated), and
isoleucine for valine (both are hydrophobic and of a similar size)
Continued..
 The substitutions that occur in proteins can also be understood with
reference to the genetic code.
For example i) Aspartate is encoded by GAU or GAC, and changing the third position to
either A or G causes the codon to encode a glutamic acid
ii) Four of the five least mutable amino acids (Trp, Cys, Phe, Tyr) are
specified by only one or two codons. A mutation of any of the three bases
of the codon is guaranteed to change that amino acid. The low mutability
of this amino acid suggests that substitution are not tolerated by natural
selection.
iii) Among the eight least mutable amino acids, only leucine is specified by
six codons, and only two (glycine and proline) are specified by four
codons.
PAM1 Matrix
 Dayhoff and colleagues used the calculated data of accepted point mutations
and the probabilities of occurrence of each amino acid to generate a mutation
probability matrix M.
Continued..
 Each element of the matrix shows the probability that an original amino acid
will be replaced by another amino acid over a defined evolutionary interval.
 This interval is one PAM, which is defined as the unit of evolutionary
divergence in which 1% of the amino acids have been changed between the
two protein sequences.
 In conclusion, the evolutionary interval of this PAM matrix is defined in
terms of percent amino acid divergence and not in units of years.
 For each original amino acid, it is easy to observe the amino acids that are
most likely to replace it if a change should occur. These data are very relevant
to pairwise sequence alignment because they will form the basis of a scoring
system in which reasonable amino acid substitutions in an alignment are
rewarded while unlikely substitutions are penalized.
 These concepts are relevant to database searching algorithms such as
BLAST which depend upon rules to score the relatedness of molecular
sequences.
Practical Usefulness of PAM Matrices in Pairwise Alignment
 Consider a pairwise alignment of two proteins; human RBP4 and
bovine -lactoglobulin and examine the outcome using the PAM40
versus the PAM250 matrix.
 The web-based SIM alignment program is suitable for this purpose.
Tools: http://www.expasy.ch
http://www.expasy.ch/tools/sim-prot.html
 PAM250 matrix is appropriate because the two proteins are only
distantly related. An overlap of 20 identical residues over a span of 81
amino acids is detected (24.7% identity).
Fig 3.16a
 PAM40 matrix shows the best aligned segment is only 10 amino
acids in length.
Fig. 3.16b
 Moreover, the short alignment is biologically meaningless. So,
PAM40 matrix is not appropriate for detecting distantly related protein
sequences.
BLOSUM scoring matrices
 A very common set of scoring matrices is the blocks substitution matrix
(BLOSUM) series.
 In 1992, Henikoff and Henikoff used BLOCKS database, which consisted
of over 500 groups of local multiple alignments (blocks) of distantly related
protein sequences.
 Thus, the Henikoffs focused on conserved regions (blocks) of proteins
that are distantly related to each other.
 BLOSUM62 matrix merges all proteins in an alignment that have 62%
amino acid identity or greater into one sequence.
 If a block of RBP orthologs includes several that have 62, 80 and 95%
amino acid identity, these would all be grouped as one sequence.
 Substitution frequencies for the BLOSUM62 matrix are weighted heavily
by protein sequences having less than 62% identity.
 The BLOSUM62 matrix, which is the default scoring matrix used by most
BLAST algorithms.
Continued..
 Henikoff and Henikoff tested the ability of a series of BLOSUM and
PAM matrices to detect proteins in BLAST searches of databases.
 They found that BLOSUM62 perform slightly better than BLOSUM60
or BLOSUM70 and dramatically better than PAM matrices at identifying
various proteins
 In fact, PAM matrices are based on data from the alignment of closely
related protein families, and they involve the assumption that substitution
probabilities for highly related proteins (e.g., PAM10) can be extrapolated
to probabilities for distantly related proteins (e.g., PAM250). In contrast,
BLOSUM matrices are based on empirical observations of more distantly
related protein alignments.
Pairwise Alignment and Limits of Detection
 If we compare human and trout RBPs, it is very easy to see their
close relationship.
 However, when we compare human RBP4 to bovine lactoglobulin, the relationship is much less obvious. Intuitively, at
some point two homologous proteins are too divergent to be
significantly aligned.
 The useful detection limits of pairwise sequence alignment can be
explored by comparing the percent identity of the two sequences
versus their evolutionary distance.
 Consider two protein sequences, each 100 amino acids in length, in
which one sequence is fixed and various numbers of mutations are
introduced into the other sequence.
 A plot of the two diverging sequences has the form of a negative
exponential.
 If the two sequences have 100% amino acid identity, they have zero
changes per 100 residues.
 If they share 50% amino acid identity, they have sustained an
average of 80 changes per 100 residues. One might have expected 50
changes per 100 residues in the case of two proteins that share 50%
amino acid identity. However, any position can be subject to multiple
hits.
 Thus, percent identity is not an exact indicator of the number of
mutations that have occurred across a protein sequence.
Continued..
 When a protein sustains about 250 hits per 100 amino acids, it may
have about 20% identity with the original protein, and it can still be
recognizable as significantly related.
 If a protein sustains 360 changes per 100 residues, it evolves to a
point at which the two proteins share about 15% amino acid identity
and are no longer recognizable as significantly related.
 The PAM250 matrix assumes the occurrence of 250 point mutations
per 100 amino acids. This corresponds to the Twilight Zone. At this
level of divergence, it is usually difficult to assess whether the two
proteins are homologous.
 In this case, multiple sequence alignment and structural predictions
are sometimes useful to assess homology in these cases.
Tests for statistical significance of Pairwise Alignments
 If two proteins share limited amino acid identity (e.g., 20-25%), it is
needed to determine whether they are significantly related.
 Alignment algorithms report the score of a pairwise alignment or the
score of the best alignments of a query sequence against an entire database
of sequences.
 Statistical tests do decide whether the matches are true positives (i.e.,
whether the two aligned proteins are genuinely homologous) or whether
they are false positives (i.e., whether they have been aligned by the
algorithm by chance).
 A main goal of alignment algorithms is thus to maximize the sensitivity
and specificity of sequence alignments.
 Sensitivity is the number of true positives divided by the sum of truepositive and false-negative results. This is a measure of the ability of an
algorithm to correctly identify genuinely related sequences.
 Specificity is the number of true negative results divided by the sum of
true-negative and false positive results. This describes the sequence
alignments that are not homologous.
Fig: Statistical analysis to find significant alignments
Significance of pairwise alignments
 A rule of thumb is that if two proteins share 25% or more amino acid identity
over a span of 150 or more amino acids, they are probably significantly related.
If we consider an alignment of just 70 amino acids, it is popular to consider the
two sequences significantly related if they share 25% amino acid identity.
 In 1998, Brenner et al., have shown that this may be erroneous, because the
enormous size of today’s molecular sequence databases increases the likelyhood
that such alignments occur by chance. For an alignment of 70 amino acid
residues, 40% amino acid identity is a reasonable threshold to estimate that two
proteins are homolgous.
 If two proteins share about 20-25% identity over a reasonable long stretch
(e.g., 70-100 amino acid residues), they are in the “twilight zone”, and it is more
difficult to be sure.
 Two proteins that are completely unrelated often share about 15-20% identity
when aligned. This is especially true because the insertion of gaps can greatly
improve the alignment of any two sequences.
Statistical significance of global alignments
A z-score (a standard score) indicates how many standard deviations
an element is from the mean. A Z score is calculated as:
Z=
x-

Where x is the current score of two aligned sequences,  is the mean
score of many sequence comparisons using scrambled sequence, and 
is the standard deviation of those measurements obtained with random
sequences.
If we need to test the alignment of RBP4 to -lactoglobulin, we first
need to align them and obtain a score. We can then scramble the lactoglobulin sequence 100 times, perform 100 alignments, record the
scores and calculate standard deviations.
If 100 alignments of shuffled proteins all have a score less than the
authentic score of RBP4 and -lactoglobulin, this indicates that the
probability (p) value less than 0.01.
Pairwise alignment tools
1) GAP – From the Genetics Computer Group (GCG)
URL: http://www.gcg.com
2) BLAST2 sequences – AT NCBI
URL: http://www.ncbi.nlm.nih.gov/BLAST/
3) Pairwise – Two Sequence Alignment Tool
URL: http://informagen.com/Applets/Pairwise/
4) SIM – Alignment tool for protein sequences from ExPaSy
URL: http://www.expasy.ch/tools/sim-prot.html
PSI-BLAST
Position-Specific Iterated BLAST
 A position specific scoring matrix, PSSM, is constructed by
calculating position specific scores for each position in the
alignment of a multiple alignment
 In the highest scoring hits in an initial blast search the PSS is
calculated by assigning high scores to highly conserved
positions and near zero scores to weakly conserved positions
 The profile is then used to perform a second BLAST search
and the results of each ‘iteration’ is used to refine the profile.
 Thus, PSI-BLAST is highly sensitive homology search
program generally used with a query of amino acid sequence
against an amino acid sequence database.
BLASTP and PSI-BLAST
 PSI-BLAST can beat BLASTP if BLASTP finds some
reliable alignments to database sequence
 PSI-BLAST can determine the positions in the query
sequence that are conserved during evolution and devise an
appropriate position-specific scoring matrix which can be
used to identify relatives at a further evolutionary distance
 If a BLASTP run can’t find any reliable alignment, PSIBLAST is powerless.
Advantages of PSI-BLAST
 PSI-BLAST offers exciting opportunities to discover new types of
relationships in protein databases and use them to infer evolutionary origins
 PSI-BLAST will search a protein sequence database with a query
sequence motif, a matrix with rows representing sequence positions and
columns representing variations in that position.
 Three advantages –There are some differences between the motifs found
by PSI-BLAST
1. The motif covers the entire sequence length in PSI-BLAST, whereas
motifs usually cover only a short stretch of the sequences
2. The same gap penalties are used throughout the procedure and there is no
position specific penalty as in other programs
3. Each subsequent motif is based on using the query sequence as a master
template to produce a multiple sequence alignment of the same length as the
query sequence
Limitations of PSI-BLAST
 The motif found by a PSI-BLAST may be evidence of structural or
evolutionary relationships but they could also be due to matching of random
variations that have no common origin or function.
 Protein structures are comprised of a tightly packed core and outside loops.
Amino acid substitution within the core are common but only certain
substitutions will work at a given amino acid position in a given structure.
Thus, sequence similarity is not usually a good indicator of structural
similarity and the motifs found need to be carefully evaluated before any firm
conclusions can be drawn.
 PSI-BLAST follows a type of algorithm called Greedy Algorithm. Once
additional sequences that match the query are found, they influence the finding
of more sequences like themselves and so on. If a different set of query
sequences were initially used, a different group with the possible overlaps with
the first set may be found. Thus, there is no guarantee that the group finally
discovered authentically represents a functional group.
Phylogenetic tree/ Dendogram
 A phylogenetic tree or evolutionary tree is a
branching diagram or tree showing the inferred
evolutionary relationships among various
biological species or other entities based upon
similarities and differences in their physical and/or
genetic characteristics.
 The taxa joined together in the tree are implied to
have descended from a common ancestor.
 The edge lengths in some trees interpreted as
time estimates.
 Each node is called a taxonomic unit. Internal
nodes are generally called hypothetical taxonomic
units (HTUs) as they cannot be directly observed.
Mutation in evolution
Distance matrix
Distance-matrix methods of phylogenetic analysis
explicitly rely on a measure of "genetic distance" between
the sequences being classified, and therefore they require
an MSA (multiple sequence alignment) as an input.
Distance is often defined as the fraction of mismatches at
aligned positions, with gaps either ignored or counted as
mismatches.
Feng and Doolittle’s progressive sequence alignment
 The most commonly used algorithm that produce multiple
alignments are derived from the progressive alignment method.
 In 1987, Da-Fei Feng and Russell Doolittle proposed this model.
 It is called progressive because the strategy entails calculating
pairwise sequence alignment scores between all proteins or DNA
sequences being aligned.
 The alignment begins with the two closest sequences an
progressively adding more sequence to the alignment.
 Feng and Doolittle’s progressive alignment occur in 3 stages.
Stage 1: The global alignment approach of Needleman and Wunsch is used to
create pairwise alignments of every protein that is to be included in a multiple
sequence alignment. As shown in the figure, for an alignment of 5 sequences,
10 pairwise alignment are generated.
Algorithm that perform pairwise alignment generate raw similarity scores.
Stage 2:
- A guide tree is calculated from the similarity or distance matrix.
- There are two principle way to construct a guide tree; the unweighted pair
group method of arithmetic averages (UPGMA) and the neighbor joining
method.
- The two main features of a tree are it’s topology (branching order) and
branch lengths (which are proportional to evolutionary distance).
- Thus the tree reflects the relatedness of all proteins to be multiply aligned.
Stage 3:
-The multiple sequence alignment is created in a series of steps based
on the order presented in the guide tree.
-The algorithm first selects the 2 most close related sequences from
the guide tree and creates a pairwise alignment.
-The next sequence is either added to the pairwise alignment or used
in another pairwise alignment.
- This procedure is continued progressively until a full alignment is
obtained.
Hidden Markov model
 Hidden Markov Models (HMMs) provide a powerful tools for
alignment.
 HMMs are the probabilistic models which describe the likelihood
that any amino acid residue occurs at each given position of an
alignment.
 A profile HMM can convert a multiple sequence alignment into a
position specific scoring system.
 A common application of profile HMMs is the query of a single
protein sequence of interest against a database of profile HMMs.
 Another application is to use a profile HMM as the query in
database search.
GTWYA
GLWYA
GRWYE
GTWYE
GEWFS
 Consider the 5 amino acid residues in
the conserved GXW region of 5
lipocalins.
- An HMM can be calculated by
estimating the probability of occurrence
of each amino acid in the 5 positions.
-In this senses the HMM approach
resembles the Position Specific Scoring
Metrics (PSSM) calculation of PSIBLAST.
-From HMM probabilities, a score can be
derived for the occurrence any specific
pattern of a related query such as,
GEWYE.
- The HMM is a model that can
described in terms of states at each
position of a sequence.
Continued..
 A profile HMM is more complex than PSSM.
 It is constructed from an initial multiple sequence alignment to define a set
of probabilities.
 Along the bottom row is a series of main states (from ‘begin’ to m1-m5 then
‘end’). These states might correspond to residues of an amino acid sequence
such as GTWYA.
 The sequence row consists of insert states (i1-i5). This states model variable
regions in the alignment, allowing sequences to be inserted as necessary.
The third row, at the top consists of circles called delete sates. This
corresponds to gaps.
They provide a path to skip a column in the multiple sequence alignment.
 Overall the protein sequence of an HMM is defined by a series of
states that are connected to each other by state transitions.
 Each state has a symbol emission probability distribution for
matching a particular amino acid residue.
 The symbol sequence of an HMM is an observed sequence that
resembles a consensus for the multiple sequence alignment.
 There are also state sequences that describe the path followed along
the Markov chain.
Collecting sequences from PSI-BLAST
Copy the sequences in MS-word
Multiple sequence alignment using CLUSTALW
(http://align.genome.jp/)
Output for PCDA (protein) by CLUSTALW
Multiple sequence alignment using TreeTop
(http://www.genebee.msu.su/services/phtree_reduced.html)
What is the number???100 or 81
Output for PCDA (protein) by CLUSTALW