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MacDNAsis was used to analyze the cDNA sequence of Troponin C (TnC) from the chicken, Gallus gallus. Using this sequence analysis, the computer program was able to predict the open reading frame for the sequence (Fig. 1).
|Figure 1. A sequence analysis of Gallus gallus cDNA for TnC. Red triangles mark start codons in the sequence, green lines indicate stop codons, and the white portions are open reading frames. The largest open reading frame (bases 1-504) is indicated in blue.|
This translated open reading frame produces a protein of 168 amino acids with a molecular weight of 18936.20 daltons.
The Kyte-Doolittle analysis is able to analyze the hydrophobicity or hydrophilicity of a protein given amino acid (AA) sequence, and predict whether that protein is a trans-membrane protein. A threshold hydrophobicity value of 1.8 is suggestive of a transmembrane protein.
|Figure 2. A Kyte and Doolittle Hydropathy analysis showing the hydrophobic (positive values) and hydrophilic (negative values) regions of the168 AA protein from the ORF of Gallus gallus TnC. There are two strongly hydrophobic regions along TnC which suggest a possible trans-membrane domain.|
The Kyte-Doolittle analysis shos that there are two domains within the protein that have a hyrdophobicity of >1.8. However, since TnC is part of the troponin complex within the cell and binds to Troponin I and Myosin, TnC is probably not a transmembrane protein.
Hopp and Woods Analysis
The Hopp and Woods analysis is another computer-based protein analysis which looks at hydrophobicity and hydrophilicity. Unlike Kyte-Doolittle, Hopp and Woods can be used to determine the antigenicity of a protein: to what portion of the protein an antibody will bind (the epitope). Usually, the most hydrophobic regions (positive values) serve as the best epitopes.
|Figure 3. A Hopp and Woods hydrophobicity test also showing hydophilic (positive values) and hydrophobic (negative values) regions of the 168 AA protein. The most hydrophilic peaks can be used as possible domains for determining an antigen.|
In TnC, there are five domains of a hydrophobicity of >2, which would probably make good epitope sites for and antibody.
Knowing the primary structure of a protein allows computer programs to predict the probably secondary structure of the protein, taking into account the different properties of the individual AAs or domains along the sequence. The secondary structrue consists of a combination of sheets, helices, turns, and coils. The secondary structure of TnC (seen in Fig. 4a and 4b) were predicted using the Chou, Fasman and Rose analysis.
|Figure 4. Predicted secondary structures of the 168 AA protein. Figure 4a shows the AA letter code and what folding corresponds to each AA in the protein (assuming it is involved in folding). Figure 4b shows a more general picture of the predicted secondary folding of TnC.|
Figure 4a and 4b show that the secondary structure of TnC is comprised of nine helices, six sheets, four turns, and six coils.
MacDNAsis is also able to simultaneously compare the protein sequences of several different proteins. Figure 5 shows a Waterman protein sequence comparison of TnC between Caenorhabiditis elegans, Drosophila silvestris, Gallus gallus (cardiac TnC), Xenopus laevis (cardiac and skeletal TnC), and Homo sapiens (cardiac and skeletal).
|Figure 5. An amino acid sequence alignment using a Waterman analysis (MacDNAsis). While there is variation between the different sequences, the chicken (Gallus gallus), frog (Xenopus laevis), and human (Homo sapiens) all have similar sequences for their cardiac TnCs.|
The cardiac TnC sequences between G. gallus, H. sapiens, and X. laevis appear to be largely similar, with a few individual differences. The other sequences show quite a bit of variation between them.
A Higgins analysis sequence comparison allows one to predict the relative relatedness between two proteins, giving an idea of how far apart evolutionarily they are separated.
|Figure 6. A phylogeny of Troponin C (TnC) created using a Higgins multisequence analysis (MacDNAsis). The diagram shows that there is a high level of homology between the Homo sapiens, Gallus gallus, and the Xenopus laevis cardiac TnC (cTnC). The skeletal muscle TnC (skTnC) shows a lower homology, while the invertebrate TnC shows little homology to the other protein sequences.|
Given the sequence similarities seen in Figure 5, it is not suprising that there is a strong correlation between G. gallus, H. sapiens, and X. laevis cardiac TnCs. The X. laevis and H. sapiens skTnC show a lower degree of relatedness to the cTnCs (but relatively high to eachother), and the C. elegans and D. silvestris show a very low degree of relatedness.
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