Example Aligning Sequences

The original bovine a-lactalbumin protein model was constructed before the availability of efficient sequence alignment programs and computational protein modeling applications. Instead of searching for similar sequences and structures using BLAST (because it did not exist), Browne et al. considered only four different solved protein structures as templates. To explore the 

CLUSTAL X Alignments Template Group Four Templates Five Templates Pairwise 

Tinman a-lactalbumin was not included in the alignment of the initially considered templates to preserve continuity between the results discussed here and those of Browne et al. 

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An interesting example of using Kohonen maps for the analysis of protein sequences is given in a journal article by Hanke (Hanke Reich, 1996). In this application, a trained Kohonen map network was used to identify protein families, aligned sequences or segments of similar secondary structure, in a highly visual manner. 

From the examination of appropriately aligned sequences, substitution matrices can be deduced. In these matrices, a large positive score corresponds to a substitution that occurs relatively frequently, whereas a large negative score corresponds to a substitution that occurs only rarely. The Blosum-62 substitution matrix illustrated in Figure 7 9 is an example. The highest scores in this substitution matrix indicate that amino acids such as cysteine (C) and tryptophan (W) tend to be conserved more than those such as serine (S) and alanine (A). Furthermore, structurally conservative... 

Fig. 1. An example of aligned sequence in multifasta format, which may be fed to PhyloGibbs the promoter of NDT80, a gene from Saccharomyces cerevisiae, and orthologous regions from Saccharomyces bayanus and Saccharomyces mikatae.

In the last example, we used RNAalifold essentially to detect structural RNAs within long aligned sequences. The method does not, however, provide a good measure for the statistical significance of predicted consensus structures. For example, it is difficult to decide whether a predicted structure is functionally important or just incidental. 

A sequence tag from an unknown protein allows a number of further options for characterisation. Even a short stretch of amino acid sequence provides a powerful means of interrogating a protein database, and may provide a useful alternative to PMF in poor quality samples that may have peptides derived from more than one protein. More advanced database searching systems will find proteins with homologous sequences to that of the peptide tag. A good example of this type of database searching system is BLAST (Basic Local Alignment Sequence Tool), (Altschul et al., 1997). A modified form of this (MS BLAST) was used in conjunction with PMF to characterise the proteome of the yeast Pichia pastoris, whose genome has not been fully sequenced... 

Figure 10.3 Sequence information captured from the PDB illustrated for PDB complex 5hvp, HIV protease with acetyl-pepstatin. (a) The raw sequence can be used by standard sequence searching and matching tools, for example, BLAST, (b) The distance annotation permits any sequence information, for example, alignment to be focused down to the binding site sequence as defined by residues within 2,4,6, or 8 Aofthe calculated cavity, (c) PDB 3D coordinate file...

The criterion used in this example was the (20x20) identical match matrix composed of ones along the diagonal and zeros elsewhere. Other criteria can be used and are in fact generally desired [see (14) for review]. They include, over the years, the minimum base change per codon required to replace one amino acid for another (15), relative weights with which amino acids substitute in aligned sequence families (16), or some comparative residue characteristics as hydrophobicity (17). 

In the investigation of the structural and functional properties of RNA molecules, comparisons of RNA sequences are an extremely powerful method. This approach requires only a critical number of carefully aligned sequences, but hardly any experimental effort. A prime example for the success of comparative sequence analysis is the structure of the ribosomal RNAs, where the comparison of sequences from more than 3000 species (1) identified Watson-Crick interactions at the level of individual nucleotides. 

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