The computer program MacDNAsis was used to analayze the sequences of phosphofructokinse (PFK) that I obtained for the last assignment (see my Genbank web page). The results of that analysis are summarized here, through a number of figures.

Determination of Largest Open Reading Frame (ORF)

Click here to see the Homo sapiens PFK sequence
Figure 1. Largest open reading frame in Homo sapiens PFK cDNA. The three rows correspond to the three possible reading frames of the sequence. Red triangles represent start codons, and the green bars demarkate stop codons. The red numbers at the bottom-left corner of the image indicate that the longest ORF in Homo sapiens PFK cDNA sequence is from nucleotides 1274-2671. To see DNA sequences of PFK, you can link to my Genbank web page or click on the figure. By using MacDNAsis to trasnlate this ORF to an amino acid sequence, I found the predicted molecular weight of phosphofruktokinase to be 51,062.18 Daltons. According to a printed source, this protein exists as a tetramer weighing approximately 340 kilodaltons (Stryer, 1996). Apparently there is some discrepancy between the number I obtained using MacDNAsis and the protein's weight in the literature.

Hydropathy Plot

Kyte & Doolittle hydropathy plot
Figure 2. Kyte & Doolittle hydrophobicity plot of Homo sapiens PFK. The translated ORF sequence (from Fig. 1) yielded this Kyte & Doolittle hydrophobicity plot. Areas above the x-axis indicate hydrophobic portions of the protein, and areas under the line represent hydrophilic ones. Parts of the protein with values greater than 1.8 (approximated by the black line in this figure) are good candidates for membrane-spanning domains. According to this prediction, there appear to be at least 5 possible membrane-spanning regions. However, judging from the function of PFK in the cell (an enzyme in the glycolytic pathway), PFK should be a cytosolic protein (because glycolysis takes place in the cytoplasm). The hydrophobic portions of the protein might be explained as inner parts of the protein not exposed to the cytosol when PFK is in its native conformation.

Antigenicity Plot

Hopp & Woods antigenicity plot
Figure 3. Hopp & Woods hydrophobicity plot of Homo sapiens PFK. The Hopp & Woods plot differs from Kyte & Doolittle plots in that the peaks in this figure correspond to hydrophilic portions of an amino acid sequence. The highest peaks represent the most hydrophilic parts, which also tend to be antigenic because they are likely to be on the outer surface of the protein's native conformation. If one wanted to make a monoclonal antibody to a linear epitope of the protein's tertiary structure, the most hydrophilic portion of the sequence would be used. For this particular protein, the sequence from approximately amino acids 33 to 66 seems to be the most ideal epitope, although many other hydrophilic regions exist.

Predicted Secondary Structure

Click here to see the actual 3D structure of PFK
Figure 4. Chou, Fasman, and Rose predicted secondary structure of Homo sapiens PFK. MacDNAsis predicted that numerous alpha helices (blue) and beta pleated sheets (red) are present in PFK. There also seem to be a number of coils (black), and four turns (green). This seems to be a good prediction, because the actual 3D structure has many helices, sheets, and coils. To see for yourself, look at a 3D Rasmol image of PFK by going to my Genbank web page.

Multiple Sequence Alignment

Click here to see the entire multiple sequence alignment
Figure 5. Multiple sequence alignment of various PFK sequences. A sample of the alignment of PFK sequences from Drosphila melanogaster, Homo sapiens, Mus musculus, Rattus norvegicus, and Schistosoma mansoni (click on the organism name to see the original sequences obtained in my genbank search). Residues highlighted in black indicate matches between sequences; unhighlighted residues are not conserved between the species. The Mus musculus and Rattus norvegicus sequences seem to be very highly related. The sequences from other species show a few similarities, but do not appear to be very related to the two aforementioned sequences.

Phylogenetic Tree

Phylogenetic Tree
Figure 6. Phylogenetic tree of various PFK sequences. The phylogenetic tree serves to predict the evolutionary relatedness of the species (Mus musculus, Rattus norvegicus, Homo sapiens, Schistosoma mansoni, and Drosphila melanogaster) on the basis of the percentage of amino acids conserved between their respective PFK sequences. As predicted from Fig. 5, Mus musculus and Rattus norvegicus are extremely (98.9%) related, which is not surprising considering both organisms belong to the subfamily Murinae. The Homo sapiens sequence is the next most closely related species, which also agrees with a taxonomical assessment (all these organisms are in the class Mammalia). The two most distantly related species taxonomically, Schistosoma mansoni and Drosophila melanogaster, are also the least related in terms of sequence.

Stryer, L. Biochemistry, fourth ed. W.H. Freeman and Co., New York: 1995. p493.

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