MacDNAsis of My Favorite Protein: Pyruvate Kinase

This webpage was created as an assignment for an undergraduate course at Davidson College in Molecular Biology.

Click here to find out more about this protein and the sequence information from the Genbank Search.  After finding out the cDNA sequence of the Homo sapiens Pyruvate Kinase gene, I used MacDNAsis to translate the sequence and determine its Open Reading Frame, molecular weight, hydrophobicity, predicted secondary structure, and the extent of its relatedness to the other four species' protein structures.

Figure 1:  Area highlighted in blue corresponds to the largest Open Reading Frame of Human Pyruvate Kinase cDNA.  Each arrow represents a possible start/stop coding sequence.  Through process of elimination the longest interval between the green lines was selected and is the ORF corresponding to the protein.

Figure 2:  Analysis of Amino Acid content of Human Pyruvate Kinase.  The molecular weight of this protein is 57.9 Kbp.  The break down of how many of each amino acid contained in the protein is listed on the left with a bar graph representation on the right.

Figure 3:  Kyte and Doolittle Hydropathy plot of Human Pyruvate Kinase.  This particular figure is very useful in discovering if a particular protein has integral membrane possibilities.  Integral membrane proteins must have hydrophobic domains which span the hydrophobic interior of the membrane.  Area above 0.00 possibly corresponds to hydrophobicity when the peak reaches the 2.00 range.  This particular hydropathy plot shows that pyruvate kinase has many possible integral membrane domains and could be an integral membrane protein.  This is consistent with Pyruvate kinase's role in cellular respiration.

Figure 4:  Hopp and Woods Hydropathy plot of Human Pyruvate Kinase.

Figure 5:  Chou, Fasman and Rose prediction of the secondary protein structure of Human Pyruvate Kinase.  The key decoding the colors are listed above the figure.  Blue corresponds to an alpha helix, red and white stripes corresponds to a beta sheet, green corresponds to a turn, and black/white stars correspond to coils.

Figure 6:  Sequence alignment of five different species' Pyruvate Kinase proteins.  Black boxes indicate similarity between sequences.  Dashed lines are included as spacers to increase similarities between the proteins.
key: cat1 = Homo sapiens (click here to see DNA sequence)
       cat2 = Saccharomyces cerevisiae (click here to see DNA sequence)
       cat3 = Mus musculus (click here to see DNA sequence)
       cat4 = Drosophila melanogaster (click here to see DNA sequence)
       cat5 = Rattus norvegicus (click here to see DNA sequence)

Finally, using the MacDNAsis program, I generated a phylogenetic tree illustrating the evolutionary relationship between the five proteins.  As would be expected cat1 (human) and cat3 (mouse) are the most closely related as they are both mammals.  The similarity between the proteins dropped off quickly between human, mouse and rat and the fruitfly and yeast were by far the least related.

Figure 7: Phylogenetic Tree comparing cat1 (Homo sapiens), cat2 (Saccharomyces cerevisiae), cat3 (Mus musculus), cat4 (Drosophila melanogaster), and cat5 (Rattus norvegicus) protein.


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