* This webpage was produced as an assignment for an undergraduate course at Davidson College*
 
 

MacDNasis Results for Enolase

Enolase cDNA from 5 different species was analyzed using MacDNAsis software. The results from the analysis of cDNA from Homo sapiens is shown in the following figures.
 

I. Determination of the largest open reading frame (ORF):
 
 

              Figure 1. MacDNAsis was used to determine the largest open reading of enolase from Homo sapiens. In the image above, the red arrows indicate start codons, and the green lines represent stop codons. The area shaded in blue is the largest open reading frame of enolase. These results indicate that the largest open reading frame is located between amino acids 75-1379. To view the original enolase cDNA sequence for Homo sapiens click here.





II. Molecular weight prediction for enolase:
 
 

                Figure 2. MacDNAsis was used to determine the molecular weight of enolase. The table above shows the amino acid content of enolase when the largest open reading frame (see figure 1) was translated. The results indicate that the molecular weight for enolase is 47,265.98 Daltons.

 

III. Kyte and Doolittle hydropathy plot of enolase:
 
 

                    Figure 3. The above image is a Kyte and Doolittle hydropathy plot of enolase that was produced using MacDNAsis software. Kyte and Doolittle hydropathy plots are used to determine whether or not a protein is an integral membrane protein. The amino acid regions that are greater than 0 on the plot are hydrophobic regions, and the areas less than 0 indicate hydrophilic amino acid regions.  If a peak reaches a value of positive 2, than the protein is considered to be an integral membrane protein. When analyzing the hydropathy plot above, it appears that enolase is an integral membrane protein with approximately 7 transmembrane regions.


 

IV. Hopp and Woods antigenicity plot for enolase:
 
 


                                   Figure 4. The above image is a Hopp and Woods hydrophilicity plot of enolase. A Hopp and Woods plot is used to predict hydrophilic regions in a protein. The amino acid regions that appear to have the most positive values on the plot are the most hydrophilic regions of the protein. Knowing which amino acids are the most hydrophilic, one can determine which portion of the protein should be used to create a peptide and a monoclonal antibody against that peptide. When analyzing the antigenecity plot above, it appears that the best portions of enolase from which a peptide and a monoclonal antibody should be generated are around amino acids 30-50, towards the middle of the protein at about amino acids 250-320, or at the end of the protein that includes the amino acids 400-430 because these are the most hydrophilic portions of the protein.

V. Secondary structure of enolase:


 
 

                Figure 5. The above image is the secondary structure of enolase that was generated using MacDNasis. According to this figure, enolase is composed of 24 alpha helices, 22 beta-pleated sheets, 2 turns, and 15 coils. If you would like to view the 3-D Rasmol structure for enolase click here.

VI. Multiple amino acid sequence alignment of enolase from 5 different species:



                            Figure 6. This figure shows the amino acid sequence similarity between enolase from 5 species: Escherichia coli, Schistosoma mansoni, Homo sapiens, Gallus gallus, and Schizosaccharomyces pombe. The amino acids that are shaded in black indicate those sequences that are similar, and those amino acids that are not shaded indicate amino acids that deviate from the other species' amino acids at those portions of the protein. Because the majority of the figure is shaded, a high degree of sequence similarity exits for enolase among the 5 different species. To see the amino acid sequences generated for the 5 species using Genbank, click on one of the following species:  Escherichia coli, Schistosoma mansoni, Homo sapiens, Gallus gallus, Schizosaccharomyces pombe.

VII. Phylogenetic tree for enolase:



                                       Figure 7. The above figure is a phylogenetic tree that shows the degree of amino acid conservation for enolase between 5 different species.  The highest degree of conservation occurs between chickens, Gallus gallus, and humans, Homo sapiens, for the phylogenetic tree indicates that these two species have 78.1% sequence similarity.  There is also a high degree of amino acid conservation in Schistosoma mansoni, which shares 64.1% sequence similarity, as well as yeast, Schizosaccharomyces pombe, which displays 52.0% sequence conservation with the other species. The greatest degree of sequence divergence occurs in E. coli, for it shares 38.0% sequence similarity. FRom this phylogenetic tree, it appears that the amino acid sequence for enolase has been highly conserved throughout evolution, for the five species indicated above so show a high degree of sequence similarity-only a slight amount of divergence is shown. To see the original amino acid sequences for the 5 species in the phylogenetic tree that were generated using Genbank, click on one of the following species:
 Escherichia coli, Schistosoma mansoni, Homo sapiens, Gallus gallus, Schizosaccharomyces pombe.
 

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