Coding
"H"

Part:BBa_K1080001

Designed by: Macquarie University   Group: iGEM13_Macquarie_Australia   (2013-09-22)

ChlH


Magnesium chelatase subunit H - chloroplast precursor; Chlamydomonas mutants with defects in this protein are chl1 and brs-1 and result in a brown phenotype [PMID: 11713666; 4436384]. Orthologous to the bacterial protein BchH [PMID: 9359397]; binds protoporphyrin and is acted upon by the ChlI:ChlD complex for magnesium insertion [PMID: 11469861]; interacts with GUN4 and may be involved in chloroplast signalling: Gene is also known as GUN5 in Arabidopsis thaliana [PMID: 11172074; 12574634]; transcript is light regulated and may be diurnal and/or circadian regulated [PMID: 16228385].

Assembly

Macquarie University 2013 iGEM Team unsuccessfully attempted assembly of this gene by Gibson Assembly:

G-Block –1 (470bp) + PCR-1 (304bp) + G-Block-2 (499bp) + G-Block-3 (499bp) + PCR-2 (984bp) + G-Block-4 (500bp) + G-Block-5 (481bp) + PCR-3 (673bp)

2013 team reported unsuccessful Gibson Assembly.


The Macquarie 2014 team is currently attempting assembly by Gibson Assembly, creating two halves with the center PCR2 element yet to connect these elements.

Part A: G-Block –1 (470bp) + PCR-1 (304bp) + G-Block-2 (499bp) + G-Block-3 (499bp)

Part B: PCR-2 (984bp)

Part C: G-Block-4 (500bp) + G-Block-5 (481bp) + G-Block 6 (673BP)


Parts yet to be fully assembled into ChlH biobrick.

Usage and Biology

Subunit of Magnesium chelatase, alongside chlD and chli1.

A structure for the analagous protein bcHH, exhibiting similar enzymatic activity, can be used as a model for chlH. This image is a three-dimensional reconstruction of the BchH·Proto complex. A, three views of the BchH·Proto reconstruction. Scale bar corresponds to 100 Å. B, three views of the apo-BchH reconstruction aligned with the BchH·Proto views in A. Note the conformational change in the thumb and finger subdomains that are fused in the BchH·Proto reconstruction (red) compared with the apo reconstruction (yellow). [http://www.ncbi.nlm.nih.gov/pubmed/18263581]


BchH.jpg
Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1928
    Illegal BglII site found at 2234
    Illegal BglII site found at 3620
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 486
    Illegal NgoMIV site found at 2152
    Illegal AgeI site found at 1132
    Illegal AgeI site found at 2650
    Illegal AgeI site found at 2704
    Illegal AgeI site found at 2923
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2254
    Illegal BsaI.rc site found at 3049
    Illegal SapI.rc site found at 2979




Amino Acid Sequence:

MCNVATGPRP PMTTFTGGNK GPAKQQVSLD LRDDGAGMFT STSPEMRRVV PDDVKGRVKV
KVVYVVLEAQ YQSAISAAVK NINAKNSKVC FEVVGYLLEE LRDQKNLDML KEDVASANIF
IGSLIFIEEL AEKIVEAVSP LREKLDACLI FPSMPAVMKL NKLGTFSMAQ LGQSKSVFSE
FIKSARKNND NFEEGLLKLV RTLPKVLKYL PSDKAQDAKN FVNSLQYWLG GNSDNLENLL
LNTVSNYVPA LKGVDFSVAE PTAYPDVGIW HPLASGMYED LKEYLNWYDT RKDMVFAKDA
PVIGLVLQRS HLVTGDEGHY SGVVAELESR GAKVIPVFAG GLDFSAPVKK FFYDPLGSGR
TFVDTVVSLT GFALVGGPAR QDAPKAIEAL KNLNVPYLVS LPLVFQTTEE WLDSELGVHP
VQVALQVALP ELDGAMEPIV FAGRDSNTGK SHSLPDRIAS LCARAVNWAN LRKKRNAEKK
LAVTVFSFPP DKGNVGTAAY LNVFGSIYRV LKNLQREGYD VGALPPSEED LIQSVLTQKE
AKFNSTDLHI AYKMKVDEYQ KLCPYAEALE ENWGKPPGTL NTNGQELLVY GRQYGNVFIG
VQPTFGYEGD PMRLLFSKSA SPHHGFAAYY TFLEKIFKAD AVLHFGTHGS LEFMPGKQVG
MSGVCYPDSL IGTIPNLYYY AANNPSEATI AKRRSYANTI SYLTPPAENA GLYKGLKELK
ELISSYQGMR ESGRAEQICA TIIETAKLCN LDRDVTLPDA DAKDLTMDMR DSVVGQVYRK
LMEIESRLLP CGLHVVGCPP TAEEAVATLV NIAELDRPDN NPPIKGMPGI LARAIGRDIE
SIYSGNNKGV LADVDQLQRI TEASRTCVRE FVKDRTGLNG RIGTNWITNL LKFTGFYVDP
WVRGLQNGEF ASANREELIT LFNYLEFCLT QVVKDNELGA LVEALNGQYV EPGPGGDPIR
NPNVLPTGKN IHALDPQSIP TQAALKSARL VVDRLLDRER DNNGGKYPET IALVLWGTDN
IKTYGESLAQ VMMMVGVKPV ADALGRVNKL EVIPLEELGR PRVDVVVNCS GVFRDLFVNQ
AVENSSWSDE SQLQEMYLKR KSYAFNSDRP GAGGEMQRDV FETAMKTVDV TFQNLDSSEI
SLTDVSHYFD SDPTKLVASL RNDGRTPNAY IADTTTANAQ VRTLGETVRL DARTKLLNPK
WYEGMLASGY EGVREIQKRM TNTMGWSATS GMVDNWVYDE ANSTFIEDAA MAERLMNTNP
NSFRKLVATF LEANGRGYWD AKPEQLERLR QLYMDVEDKI EGVE


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

Number of amino acids: 1364

Molecular weight: 150738.5

Theoretical pI: 5.39

Amino acid composition:
Ala (A) 114 8.4%
Arg (R) 69 5.1%
Asn (N) 79 5.8%
Asp (D) 81 5.9%
Cys (C) 13 1.0%
Gln (Q) 46 3.4%
Glu (E) 91 6.7%
Gly (G) 100 7.3%
His (H) 14 1.0%
Ile (I) 51 3.7%
Leu (L) 141 10.3%
Lys (K) 79 5.8%
Met (M) 35 2.6%
Phe (F) 50 3.7%
Pro (P) 70 5.1%
Ser (S) 84 6.2%
Thr (T) 69 5.1%
Trp (W) 14 1.0%
Tyr (Y) 49 3.6%
Val (V) 115 8.4%
Pyl (O) 0 0.0%
Sec (U) 0 0.0%

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


Total number of negatively charged residues (Asp + Glu): 172 Total number of positively charged residues (Arg + Lys): 148

Atomic composition:

Carbon C 6703 Hydrogen H 10581 Nitrogen N 1817 Oxygen O 2036 Sulfur S 48

Formula: C6703H10581N1817O2036S48 Total number of atoms: 21185

Extinction coefficients:

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

Ext. coefficient 150760 Abs 0.1% (=1 g/l) 1.000, assuming all pairs of Cys residues form cystines


Ext. coefficient 150010 Abs 0.1% (=1 g/l) 0.995, 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 32.83 This classifies the protein as stable.


Aliphatic index: 87.71

Grand average of hydropathicity (GRAVY): -0.260

Source

Chlamydomonas reinhardtii

References

1. Sirijovski, N., Lundqvist, J., Rosenbäck, M., Elmlund, H., Al-Karadaghi, S., Willows, R.D., and Hansson, M. (2008). Substrate-binding Model of the Chlorophyll Biosynthetic Magnesium Chelatase BchH Subunit. J. Biol. Chem. 283, 11652–11660.


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