Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Multiple Sequence Alignment 233
ensure that the quality of the final product remains high at reasonable levels of
pairwise sequence similarities (>30% sequence identity). However, as the
degree of sequence similarity decreases below 25%, it generally becomes difficult
to separate a legitimate “signal,” or sequence conservation, from “noise,”
or apparent similarities between motifs that are, in fact, unrelated and should
not be matched. In-depth treatment of popular methods available for aligning
sequences is available elsewhere (e.g., ref. 14) as this is beyond the scope of
this chapter. The alignments described here are based on the dynamic programming
(DP) algorithm as implemented in the program PRALINE (14) and the
popular method CLUSTALW (15) as representative programs.
This chapter will concentrate on specific methods available for sequence
alignment of calcium-binding proteins. One particularly important question to
address in the examination of these proteins is which part of the polypeptide
chain one wishes to align and the degree of similarity shared with other protein
sequences under consideration. If the protein of interest belongs to a particular
subfamily in which the topology is roughly the same, as is the case for the
calmodulin and troponin C families, then a global alignment program would be
ideal. Global-alignment methods align input sequences over their full length
and can be confused if, for example, some sequences contain unrelated domains
in addition to the calcium-binding domain. Therefore, if sequences are being
compared of very different lengths and topologies (e.g., the entire family of
EF-hand or all C2-domain proteins) and the region of interest is only the calcium-binding
area, then a local alignment algorithm would be judicious. Both
of these situations will be addressed in this protocol. Additionally, some techniques
to create phylogenetic trees will be presented and specific results of
sequence alignments will be given in the notes to provide simple, but concrete,
examples (see Subheading 4.2.). Throughout, it will be assumed that the
researcher has access to the World Wide Web, although it is certainly possible
to perform alignments if sequence databases and alignment programs are available
locally. A compilation of Web sites holding methods and/or databases
important for this chapter is given in Table 2.
2. Materials
1. A sequence (or set of sequences) for alignment known as the target sequence
(set).
2. Access to one or more sequence databases to search against, such as SWISS-
PROT (16), EMBL (17), GENBANK (18), or PIR (19).
3. An alignment program suitable for aligning the sequences of interest such as
PRALINE, or CLUSTALX (20) (see Note 1).
4. Any additional biochemical or structural evidence known about the sequence,
such as metal-binding sites or secondary structure information.