Muhammet A Celik (Matric No.

: SES100726 )
mastercod3r@hotmail.com
Dr. Saharuddin Bin Mohamad
Current Topics in Bioinformatics
Semester I, 2014/2015

Article Summary
Evolutionary trace analysis of plant haemoglobins: implications for site-directed
mutagenesis

The evolutionary trace (ET) is the single most used approach to classify and
identify protein functional elements and targets the most related sites of a protein.
ET applies to the complete proteome; its predictions come with a reliability score;
and outcomes typically reach significance in most protein families with 20 or
more sequence homologues. In order to identify functional sites, ET scans a
multiple sequence alignment for residue variations that is linked with major
evolutionary divergences. In case studies this enables the selective separation

of

functional sites and. On a larger scale, this allows for specific function predictions
based on motifs built from selected ET-identified residues.

In this article, the scientists had used Evolutionary Trace method to identify the
potential targets for mutagenesis in plant haemoglobins with the purpose of
finding specificity determinants for symbiotic (Lbs) and non-symbiotic (nsHbs)
from plants. As with the help of Multiple Sequence Alignment, the scientists could
compare the consensus sequences for groups of proteins that are share the same
node within the phylogenetic tree which generates a trace. After that the
residue is categorized into three classes which are class-specific, absolutely
conserved and lastly neutral. Sequence conservation conducts in defined protein
family

groups

to

differentiate

features

of

symbiotic

and

non-symbiotic

haemoglobins. Then identified trace residues can be mapped on known protein

structures to determine clusters of essential amino acids.

The methodology that is used in the article

Homologous sequences were obtained with BLASTp using the SWISS PROT
protein database and aligned with ClustalW. After removal of sequences with
gaps and outliers of the sequence similarity tree, 74 sequence of the plant
haemoglobin family was obtained. Several protein weight matrices such as

Blosum 30. Blosum 45. Blosum 62, Gonnet and PAM250 were used to generate
the sequence similarity index. Then ET analysis was carried out using BLOSUM62
to allow for substitution of similar amino acids. The aligned sequences and the
1D8U, !BIN coordinates were submitted to the Cambridge University ET server
with default parameters. After constructing a phylogenetic tree, the phylogram is
divided by evolutionary time cutoff lines, into ten evenly distributed partitions
P01 to P10. After that sequences within different classes, in a given partition were
separately aligned and the resultant aligned classes were compared to derive
their consensus trace residues. Then trace residues were mapped onto known
structures of soya bean laghaemoglobin (1BINA) and non-symbiotic haemoglobin
from rice (1D8U) obtained from Protein Data Bank and visualized by pymol.

Results and Discussion:

A phylip distance matrix based on sequence identity is generated for 74 plant

haemoglobins sequence identified from SWISSPROT database by the CLUSTAL
software and then the resulting seqeunce alignment after careful examination is
submitted to the TraceSuitII server for ET anaylsis where a a rooted phylogenetic
tree is produced by Kitsch algorithm. The partitions P01 to P10 divide the
phylogenetic tree into classes that vary with the partitions. (Fig.1)