Figure 1. Search results for cat Cytochrome C Oxidase Subunit II 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 1 to base pair 137.
Figure 2. Amino acid content of the protein sequence translated from
the cat Cytochrome C Oxidase Subunit II cDNA sequence. This shows
the molecular weight as well as the concentration of amino acids.
The molecular weight of the protein sequence was calculated to be 24977.68 daltons, or about 24.98 kDa.
Figure 3. Hydropathy plot for protein sequence translated from cat
Cytochrome C Oxidase Subunit II cDNA using the Kyte and Doolittle algorithm.
A window of 6 amino acids was used and the threshold for a transmembrane
domain was 0.0.
The hydropathy plot shows that there is possibly 10 hydrophobic areas
of this subunit protein. These could be areas that are inside the
protein when it is in its tertiary structure. Since Cytochrome C
Oxidase Subunit II is a subunit, these hydrophobic areas may also be hidden
beneath the other subunits.
Figure 4. Antigenicity plot for protein sequence translated from Cat
Cytochrome C Oxidase Subunit II cDNA. The algorithm used was that
of Hopp and Woods and the window used was 6 amino acids.
The Hopp & Woods method of predicting secondary structure paints a similar picture of the protein. This shows the hydrophilic and highly charged areas of the polypeptide. There does not appear to be more than three hydrophilic and highly charged areas.
I think that a likely place to use for monoclonal antibody generation would be the areas between 55 and 61 because they are the most hydrophilic. If one wanted to just generate monoclonal antibodies for Cytochrome C Oxidase, I would suggest using a more hydrophilic subunit (I or III).
Figure 5. Secondary structure prediction for protein sequence translated
from Cat Cytochrome C Oxidase Subunit II 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.
Fig. 6. Secondary Structure Prediction
 |
The secondary structure predicted is fitting for a subunit protein. The easiest way to picture the protein is by using the Rasmol image of Cytochrome C Oxidase Subunit II. I would also recommend looking at this protein as it interacts with the other cytochrome c oxidase subunits. Rasmole image of entire Cytochrome C Oxidase. This will also help to reveal the possible reasons for the many hydrophobic regions of this subunit (such as subunit binding sites).
Figure 6. Multiple alignment results for the amino acid sequences for
Cytochrome C Oxidase Subunit II from the five genome organisms. 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 the three mamalian species and amphibian match up very closely. This indicates that cytochrome c oxidase subunit (and most likely the whole protein) has been conserved through evoultion.
I also used the MacDNAsis program to generate a phylogenetic tree based on the sequence homology of Cytochrome C Oxidase Subunit II. The tree is shown in Fig. 8.
Figure 7. Phylogenetic tree based on the sequence homology of Cytochrome
C Oxidase Subunit II for Cat, Horse, Tarsius, Frog, and Yeast. The
estimated percentage homology with the Cat amino acid sequence is indicated
on each branching point.
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Any comments or questions can be answered by e-mailing me at ankazama@davidson.edu