Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

242 Weljie and Heringa
compared, and a final file was created with all unique sequences. This included
all false-negative entries from the PROSITE database and selected ancestral
calcium-binding regions to examine the manner in which these regions have
“evolved” out of calcium binding, as well as provide a reasonably difficult test
set with sequences of low homology. Finally, these sequences were subjected
to a 0–50% identity cut-off using OBSTRUCT (see Table 3). The final set of
sequences were aligned with both PRALINE and CLUSTALX using the
BLOSUM62 matrix with gap opening and extension penalties of 12 and 1,
respectively. The resulting alignments were analyzed by creating NJ trees in
CLUSTALX, and visualizing the resultant files with the UNROOTED program.
Finally, the alignments were recreated with outlier sequences in order to establish
distant branches, and then bootstrapped trees were created. These trees
were visualized with the NJPLOT program.
4.2.2. Results
As a result of space limitations and the large size of the EF-hand and
C2-domain family alignments, representative sequences are shown here; and
the complete alignments as well as examples of the NJ trees are available via
the world wide web at http://groningen.bio.ucalgary.ca/cabp-alignments.html.
In the following text, the analysis refers to the complete alignments, and references
to representative figures will be made explicitly. Briefly, the key results
from these sets of alignments are presented below.
1. EF-hand family: Both programs aligned the key residues for this family in a
reasonable manner (see Fig. 1). The first Asp that coordinates calcium in the
EF-hands was aligned well in both programs for the sequences showing strong
canonical EF-hand characteriztics, as were the other key ion-binding residues for
most sequences. As expected, difficulties arose with sequences that were very
distantly related showing little homology, as PRALINE left the three least
homologous sequences with very little overlap to the previous sequences, and
CLUSTALX introduced large gaps in the majority of sequences to deal with the
same problem. The computations were repeated with the six least homologous
sequences removed, which greatly improved both alignments. As shown in
Fig. 1, the matrix and gap penalties used with the ClustalX algorithm resulted in
more gaps throughout the alignment, whereas PRALINE only opened a maximum
of two in any sequence. The phylogenetic trees, which were created using
these alignments were reasonably stable (see website), although many key columns
were discounted during bootstrapping, which was based only on alignment
positions without gaps since those would reduce the stability of the tree.
This could be remedied by further manual aligning of the sequences or
removing the sequences containing serious gaps that correspond to key residues
in the EF-hand pattern of the remaining sequences. Alternately, a differ-