MacDNAsis analysis of triosephosphate isomerase sequences from genome organisms

After collecting the cDNA and amino acid sequences for the five genome organisms from Genbank (see this page for details), I used the human cDNA sequence for analysis with the MacDNAsis sequence analysis software. The results for the analysis are presented below.

Open Reading Frame (ORF) search

MacDNAsis was used to search the human cDNA sequence for open reading frames. The search showed that the largest open reading frame was in the frame starting with the second base pair in the cDNa sequence, and was from base pair 263 to base pair 1117. The search results are shown in Fig. 1 below.


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.

Protein sequence and molecular weight

The MacDNAsis program was used to translate the largest open reading frame in the cDNA (see above) into the corresponding amino acid sequence, and calculate the number of occurrences of each amino acid and the molecular weight of the the protein. The results of the translation are shown in Figure 2 below.


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.

Hydropathy plot of amino acid sequence

A hydropathy plot for the translated protein sequence was done using the Kyte and Doolittle algorithm. The results are shown in Fig.3.


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.

Antigenicity analysis

An antigenicity plot was generated for the translated protein sequence using the Hopp and Woods algorithm. The plot is shown in Fig. 4.


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.

Secondary structure prediction

I used the Chou, Fasman and Rose algorithm to predict the secondary structure of the protein using the primary sequence. The results are shown in Fig.5.


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).

Multiple sequence alignment and phylogenetic tree

The Genbank amino acid sequences for triosephosphate isomerase from the five genome organisms was used to perform a multiple sequence alignment test for sequence homology. The original protein sequences can be found here - human, mouse, Drosophila, yeast and C. elegans. The results of the alignment are shown in Fig.6.


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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Comments? Questions? Suggestions? E-mail rakarnik@davidson.edu.