Electronic Lab Notebook

Week 2: Aipotu Biochemistry Lab

Specific Tasks:

What are the differences in the amino acid sequences of the proteins produced by the alleles you define in Part I? Hint: use the Compare menu to find the difference(s) between the amino acid sequences.

What features of the amino acid sequence make a protein pigmented?

What features of the amino acid sequence make a protein a particular color?

How do the colors combine to produce an overall color? How does this explain the genotype-phenotype rules you found in part (I)?

Which proteins are found in each of the four starting organisms?

Using this knowledge, construct a purple protein.

Methods

In this lab, I used Aipotu Biochemistry software to analyze the effects of amino acid sequence on flower phenotype. First, I decided to look for patterns and correlations between amino acid sequences and phenotype (that is, protein color). Using the list of 'interesting' amino acid sequences provided (see page 12), I made my initial observations. Inputting the sequences in the folding window, I took note of various features of the 2D structures formed including hydrophobic interactions (indicated by patterns of clustering of like-colored amino acid residues; the darker the amino acid, the more hydrophobic), ionic interactions, and behavior of hydrogen bond forming amino acids. To add to my initial observation set, I looked at the alleles for the proteins of the four organisms in the Greenhouse. To help aid in my observations, I used the 'Compare'>'Upper vs. Lower' function on Aipotu. This allowed me to locate changes between similar amino acid sequences. Based off these observations, I came up with my initial hypotheses for this investigation:

Proteins will be colored depending on the presence of absence of specific amino acid residues
1. Proteins with amino acid Phe will be red
2. Proteins with amino acid Trp will be yellow
3. Proteins with amino acid Tyr will be blue
Secondary colors (i.e. orange and green) are formed due to the combination of color-determining amino acids
Thus, combining two alleles that code for protein pigments Red and Blue will create a flower of Purple color.

Using this, I constructed a number of proteins consisting of the various amino acids. By constructing a colorless protein using Phe, Trp or Tyr, my hypothesis would be deemed inadequate.

Results

Observations

RED (indicated by presence of Phe?)
- Phe Phe Phe Phe Phe Phe Phe Arg Arg Arg Arg Arg Arg
  - Hydrophilic 6 member tail
  - 7 member hydrophobic Phe head
- Arg Arg Arg Phe Phe Phe Phe Phe Phe Phe Arg Arg Arg
  - 2x hydrophilic, positive 3 member tails (arginine)
  - 7 member hydrophobic Phe head
- Lys Lys Lys Lys Lys Lys Leu Leu Leu Leu Leu Leu Phe
  - hydrophilic, positive 6 member tail (lysine)
  - hydrophobic head (6leu, 1 Phe)
- TESTING
  - Phe Phe Phe Phe Phe Phe gives white
  - Tried with a hydrophilic tail (ffffffrrrrrr) - still white
  - Putting a seventh hydrophobic molecule to form the head gave Red
GREEN
- Met Ser Asn Arg His Ile Leu Leu Val Tyr Trp Arg Gln
  - 6 hydrophilic molecules toward outside (3+, 3-)
YELLOW
- Glu Glu Glu Trp Trp Trp Trp Trp Trp Trp Glu Glu Glu
  - 2 negative polar, hydrophilic tails
  - 7 member hydrophobic head (Trp)
ORANGE
- Ser Leu Gln Leu Asn Ile Thr Met Glu Val Asp Phe Trp
WHITE
- hydrophilic, positive, 6 member tail
- 7member hydrophbic Leu Head
BLUE
- Indicated by presence of Tyrinine

Starting Organism	Allele	Color	Amino Acid Sequence
Green-1	C^G	Green	Met Ser Asn Arg His Ile Leu Leu Val Tyr Trp Arg Gln Met Ser Asn Arg His Ile Leu Leu Val Tyr Trp Arg Gln
Green-2	C^BC^Y	Green	Met Ser Asn Arg His Ile Leu Leu Val Tyr Cys Arg Gln Met Ser Asn Arg His Ile Leu Leu Val Trp Cys Arg Gln
Red	C^R -	Red	Met Ser Asn Arg His Ile Leu Leu Val Phe Cys Arg Gln
White	C^WC^W	white	Met Ser Asn Arg His Ile Leu Leu Val Val Cys Arg Gln Met Ser Asn Arg His Ile Leu Leu Val Val Cys Arg Gln

What features of a protein make it colored?
1. Colored proteins must have at least 6 hydrophilic molecules, 7 hydrophobic molecules, and at least 1 of: Phe, Trp or Tyr
What features of the amino acid sequence make a protein a particular color?
1. Presence of specific amino acids determine color. Phe is red, Trp is yellow and Tyr is blue.
How do the colors combine to produce an overall color? How does this explain the genotype-phenotype rules you found it Part (I)?
1. Flowers that appear as a secondary colors (orange, purple and green) may be formed by combining two proteins of the appropriate primary color (ie. Orange is red + yellow, Purple is blue + red, and Green is yellow + blue)
2. This is in accordance with the previous findings that Orange, Purple and Green phenotypes are due to the incomplete dominance of the colored alleles
3. Furthermore, individual proteins may be a secondary color if the appropriate color determining amino acids residues are both present in the amino acid sequence of the protein
Show your TA that you have made a purple protein. For full credit, you need to explain to your TA why it is purple
1. This protein appears purple due to it's amino acid sequence. There are 6 hydrophilic molecules and 7 hydrophobic molecules, allowing it to produce color
2. The amino acid sequences also contain Phe and Tyr. These color coding alleles demonstrate incomplete dominance and thus result in the appearance of a 'blended' red and blue phenotype. This provides the flower with its purple color.

Conclusion

Through the Aipotu Biochemistry Lab, the role of amino acid sequence in determining phenotype was examined. In the model presented, a minimum of 6 hydrophilic and 7 hydrophobic amino acids were necessary for the protein to exhibit color. The presence of specific amino acids determined protein color. Phe resulted in a red protein, Trp in a yellow protein and Tyr in a blue protein. This is perhaps due to the aromatic structure of these amino acids, as the increased conjugation allows for the absorption of light and reflection of color. These alleles were dominant over the white phenotype, but exhibited incomplete or codominance to each other. Combining a protein with the red allele and yellow allele resulted in an orange phenotype, while yellow and blue resulted in a green phenotype and red and blue resulted in a purple phenotype. Furthermore, these color-coding amino acids (Phe, Trp and Tyr) could be combined in a single amino acid sequence to code for a colored protein.

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