Figure 1. Search results for open reading frames using the MacDNAsis program.Standard start and stop codons were used for the search. The red inverted triangles represent start codons and the vertical green lines represent the stop codons. The blue area marks the largest open reading frame found, from base pair 263 to base pair 1117.
Figure 2. Amino acid content of the protein sequence translated from the cDNA sequence. Note the molecular weight calculation of the protein.
The molecular weight of the protein sequence was calculated to be 30534.05 daltons, or about 30.5 kDa.
Figure 3. Hydropathy plot for protein sequence (translated from human triosephosphate isomerase cDNA) using the Kyte and Doolittle algorithm. A window of 8 amino acids was used and the threshold for a transmembrane domain was 1.8.
The hydropathy plot shows that there is only one peak that reaches the threshold of 1.8 used to determine the existence of a transmembrane domain. Triosephosphate isomerase is involved in glycolysis which takes place in the cytoplasm of a cell, and should not be a integral membrane protein. Therefore the region of high hydrophobicity is probably just one that is on the inner side of the tertiary structure, and does not really indicate a transmembrane region. We must remember that the Kyte and Doolittle algorithm is merely a computer prediction of tertiary structure, and may not be correct.
Figure 4. Antigenicity plot for protein sequence (translated from human triosephosphate isomerase cDNA). The algorithm used was that of Hopp and Woods and the window used was 8 amino acids.
The antigenicity plot is an indication of the areas of the protein that are hydrophilic and highly charged, making it more likely that these regions would be on the outside of a tertiary structure. Such charged regions of the protein would be the ones most easily used as epitopes for antibodies the protein. For our protein sequence we see that there are several promising regions, but the best choices are either the N-terminus or a region between the amino acids 165-180. These regions are most likely to be "sticking out" out of the protein tertiary structure, and would be those best accessible to an antibody molecule.
Figure 5. Secondary structure prediction for protein sequence translated from human triosephosphate isomerase cDNA. The algorithm used was that of Chou, Fasman and Rose. The "H" strings mark helical structure, the "S" strings mark sheets, the "t"s mark turns and the "C"s mark coils.
The secondary structure predicted is typical of a globular protein, with a good mixture of helical coils and pleated sheets. This is expected for an enzyme like triosephosphate isomerase and can be seen in the Rasmol image of triosephosphate isomerase (MMDB Id: 2490, PDB Id: 1YPI).
Figure 6. Multiple alignment results for the amino acid sequences for triosephosphate isomerase from the five genome organisms - human (timhum.aa), yeast (timsac.aa), mouse (timmus.aa), Drosophila (timdro.aa) and C.elegans (timcel.aa). The consensus sequences are highlighted in black.
The multiple sequence alignment results show that a large proportion of the amino acids in the primary sequence of triosephosphate isomerase match up. This probably indicates that this protein has been highly conserved through evoultion, since it plays such an important role in metabolism.
I also used the MacDNAsis program to generate a phylogenetic tree based on the sequence homology of triosephosphate isomerase. The tree is shown in Fig. 7.
Figure 7. Phylogenetic tree based on the sequence homology of triosephosphate isomerase for the five genome organisms - human (timhum.aa), yeast (timsac.aa), mouse (timmus.aa), Drosophila (timdro.aa) and C.elegans (timcel.aa). The percentage homology with the human amino acid sequence is indicated on each branching point.
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