Coding
"Plasto"

Part:BBa_K1080006

Designed by: Macquarie University   Group: iGEM13_Macquarie_Australia   (2013-09-12)

Plastocyanin


Chloroplast precursor - Plastocyanin contains copper and is a chloroplast precursor protein. It is taken up after post translation and placed on its functional site where it is involved in electron transfer between cytochrome f of the cytochrome b6f complex from photosystem II and P700+ from photosystem I.

Operon Usage

PSB1C3 Operon 2.png
This gene has been used in an operon with other genes responsible for catalysing the biosynthesis pathway from Mg-protoporphyrin IX to Protochlorophyllide. CTH1, Plastocyanin, and YCF54 are involved in the oxidative cyclase pathway. ChlM methylates Mg-protoporphyrin IX, facilitating the highly-regulated catalysis of Mg-chelatase. CTH1 catalyses the conversion of Mg-protoporphyrin IX monomethyl into divinyl protochlorophyllide, interacting with YCF54 and Plastocyanin.
The insertion of magnesium is the key component of the chlorophyll biosynthesis pathway.
The plasmid is under the control of the lac promoter.

Structure


                                  Plastocyanin.jpg
     Figure 1. Structure of Plastocyanin with emphasis on the copper binding site and the Tyr-83 side chain.

Shown above is a structure of plastocyanin with emphasis on the tetrahedral structured copper binding site which is located at the "north" end of the molecule. The Tyr-83 side chain is also shown on the "east" end of the molecule also known as the "remote" site of electron transfer.



Biobrick Design:

Source Genbank accession: [http://www.ncbi.nlm.nih.gov/protein/AAA32834.1 AAA32834.1]



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 100
    Illegal BsaI.rc site found at 223


Characterisation of Plastocyanin


Our tac+plastocyanin composite biobrick theoretically expresses a copper containing protein. Colonies containing this biobrick should turn blue in the presence of inducer (IPTG) and copper. To test this, we plated this culture out on a regular LB plate, and a copper + IPTG LB plate. We also plated out a control plate with both Plasto and CHLI1 expressing colonies.

The first image below show plastocyanin expressing colonies growing blue on LB+copper+IPTG plates seen on the right, and white/yellow on regular LB plates shown on the left:



The second image shows a LB+copper+IPTG plate with plastocyanin colonies growing on one half, and non-expressing colonies (ChlI1) on the other half:





Ribbon diagram of protein structure

http://pfam.sanger.ac.uk/structure/getimage?id=2plt

Amino acid sequence

MATVKLGADS GALEFVPKTL TIKSGETVNF VNNAGFPHNI VFDEDAIPSG VNADAISRDD
YLNAPGETYS VKLTAAGEYG YYCEPHQGAG MVGKIIVQ

References and documentation are available. Please note the modified algorithm for extinction coefficient.

Number of amino acids: 98

Molecular weight: 10339.5

Theoretical pI: 4.57

Amino acid composition: Ala(A) 11 ( 11.2% )
Arg(R) 01 ( 1.0% )
Asn(N) 06 ( 6.1% )
Asp(D) 06 ( 6.1% )
Cys(C) 01 ( 1.0% )
Gln(Q) 02 ( 2.0% )
Glu(E) 06 ( 6.1% )
Gly(G) 11 ( 11.2% )
His(H) 02 ( 2.0% )
Ile(I) 06 ( 6.1% )
Leu(L) 05 ( 5.1% )
Lys(K) 05 ( 5.1% )
Met(M) 02 ( 2.0% )
Phe(F) 04 ( 4.1% )
Pro(P) 05 ( 5.1% )
Ser(S) 05 ( 5.1% )
Thr(T) 06 ( 6.1% )
Trp(W) 00 ( 0.0% )
Tyr(Y) 05 ( 5.1% )
Val(V) 09 ( 9.2% )
Pyl(O) 00 ( 0.0% )
Sec(U) 00 ( 0.0% )
</FONT> 

(B)   0	  0.0%
(Z)   0	  0.0%
(X)   0	  0.0%


Total number of negatively charged residues (Asp + Glu): 12 Total number of positively charged residues (Arg + Lys): 6

Atomic composition:

Carbon C 460 Hydrogen H 708 Nitrogen N 118 Oxygen O 147 Sulfur S 3

Formula: C460H708N118O147S3 Total number of atoms: 1436

Extinction coefficients:

This protein does not contain any Trp residues. Experience shows that this could result in more than 10% error in the computed extinction coefficient.

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.

Ext. coefficient 7450 Abs 0.1% (=1 g/l) 0.721, assuming all pairs of Cys residues form cystines


Ext. coefficient 7450 Abs 0.1% (=1 g/l) 0.721, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is M (Met).

The estimated half-life is: 

                            30 hours (mammalian reticulocytes, in vitro).
                           >20 hours (yeast, in vivo).
                           >10 hours (Escherichia coli, in vivo).


Instability index:

The instability index (II) is computed to be 18.58 This classifies the protein as stable.


Aliphatic index: 81.63

Grand average of hydropathicity (GRAVY): -0.065

Source

From genome of Chlamydomonas reinhardtii

References

Redinbo, M.R., et al. (1994). "Plastocyanin: structural and functional analysis". Journal of bioenergetics and biomembranes. Vol. 26, No.1


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