Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Multiple Sequence Alignment 241
Table 3
Summary of Sequence Retrieval Statistics for Calcium-Binding
Domains (Number of Sequences)
Pfam PROSITE After 50% cut-off
EF-hand 790 479 188
C2 domain 109 50 60
Annexin 171 54 37
tiple alignment, although it must be kept in mind that the biological significance
of a particular tree is not addressed.
A researcher wishing to derive a phylogeny from a given set of protein
sequences, should first seek one or more sensitive multiple alignment routines to
construct a good alignment. If secondary or tertiary structures are known, the
information should be applied to check and enhance the alignment. A variety of
phylogenetic methods should then be used (preferably including the NJ method)
in conjunction with bootstrapping and the results compared carefully for consistency.
Paradoxally, many wrong trees that can be derived from a particular sequence
set will lead to far more interesting phylogenies than the one correct tree.
4.2. Specific Practical Examples Using Calcium-Binding Proteins
In order to provide some practical insight into the methodology outlined
above, example alignments of the EF hand, C2 domain, and annexin families
are provided below. The results presented are not intended to provide conclusive
results of any sort, but to demonstrate a general methodology, simple
interpretations of results, possible pitfalls and ways to avoid them. The
emphasis is on alignment of sequences with large identity differences, and on
contrasting the performance of different alignment methods (exemplified here
by the PRALINE and CLUSTALX algorithms) in the alignment of the various
families.
4.2.1. Methodology
The basic procedure used was as described in Subheading 3., and this section
is included to provide some example of the scope and procedures involved
in these alignments. Sequences were extracted from the PROSITE and PFAM
databases using the terms “EF hand,” “C2,” and “annexin” via the SRS interface.
Table 3 provides a summary of the number of sequences available from
each. Because of the variant nature in sequence length and the presence of
repeats, it was decided that the alignments should be performed for the calciumbinding
regions only, with each calcium-binding site having its own sequence
entry. For each of the alignments, the sequences in these two databases were