Coiled-coil protein prediction. It is important to get students to think of sequence analysis as a kind of experimental process. In particular, get them to do “experiments” with the appropriate controls to test the algorithms. One particular algorithm for the prediction of coiled coil-forming segments in proteins from amino acid sequences (Lupas et al., 1994) was tested using a variety of proteins with known structures that either contained or lacked coiled coils. After testing the algorithm (www.ch.embnet.org/software/COILS_form.html) with positive and negative “control” proteins, the algorithm was then tested on an “unknown” protein. As examples of positive controls, students used tropomyosin and myosin protein sequences. As examples of negative controls, students used the sequences for an immunoglobulin domain (β-sheet structure) and a hemoglobin subunit (α-helical but lacking coiled coils). Finally, they compared the prediction obtained from α- or β-tubulin sequences (noted in Lupas et al., 1994) with the crystal structure of the tubulin dimer (1tub.pdb) using the RasMol program. Identification of EF-hand Ca2+-binding protein motifs. Pencil-and-paper exercises first trained students to identify the presence of a protein motif common to calcium-binding proteins such as calmodulin and tropomyosin. The students were then given the sequences of two actin cross-linking proteins: human L-plastin (shows Ca2+-regulated cross-linking of actin filaments) and yeast fimbrin (a plastin homologue). They were asked whether or not they would predict that the actin cross-linking activity of yeast fimbrin was likely to be regulated by calcium. Phylogenetic trees derived by comparison of globin protein sequences. Students were given numbered but otherwise unlabeled proteins sequences for globins from a diverse set of organisms. Students carried out a multiple sequence alignment using ClustalW, followed by the generation of a phylogenetic tree using TreeView. Students were then given a key indicating the identity of the organism from which each of the numbered globin sequences was derived. They were asked whether the phylogenetic tree they generated was consistent with our understanding of the evolutionary relationships of these organisms.
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Modular organization of titin. Students examined the modular organization of this large protein, with an emphasis on structure of the numerous type I and II repeats found in this protein. Sequence and structural analysis of common protein modules. Students carried out a comparative study of one of several common protein modules or domains. Examples include SH2, SH3, pleckstrin homology (PH), calponin homology (CH), and WD or WD-like domains. -
Sequence and structural analysis of specific components of complex biological assemblies. Students looked at the structure and function of specific protein components of cellular or viral machines such as the proteasome, chaperonin, ATP synthase, and picornavirus capsids.
Software required: Web Browser, RasMol, TreeView
Useful Web sites: BLAST @ NCBI, Clustal W @ EBI, RCSB, COILS, VAST
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