Max -Rubisco Flashcards
what does rubisco stand for
ribulose-5-bisphospahte carboxylase oxygenase
Rubisco is amde up of
8 large and 8 small subunits
why are chaperones required
in vivo protein folding is very crowded requires assistance- molecular chaperones
chaperones involved in protein folding
Bsd2
Cpn 60
Cpn10
Cpn20
chaperones involved in protein assembly
RbcX
Raf1
Raf2
RbcL c terminus???????
engineer pants rubisco in whar 3 ways
- change aa (catalytic switches)
- swap rubisco
- swap rubisco + chaperones
example of changes in aa in rubisco
2-5 aa replacement in rbcL gene by homologous recombination resulted in kinetic changes in tobacco plants
example of swap rubisco
tobacco plant
substitute rbcL gene in chloroplasts +AB res gene via homologous recombination
- potato similar growth to WT, not better
- other rubiscos, cause plants not to grow - rubisco had not folded correctly - incorrect chaperones
greater the reduction in growth the greater the aa difference
how do small su enter chloroplasts
cytosol nuclear genome rbcs gene . signal peptide directs proteni into chlorplast via tictoc complex
other chaperones also enter via tictoc complex
(large su + otehr chapeones encoded for in chlorplast)
Cpn60 homolog?
isoforms?
GroEL
Cpn60 has two isoforms dont know which one is used
Cpn10-20 homolog?
GroES
Raf1 stands for? discovery?
Rubisco accumulation factor 1
discovered recently 2017 - by generation of random mutations by gamma rays
- rubisco was expressed but didnt function
experiment by Whitney et al 2015 did what?
assembled foreign rubisco w/ native Raf1 gene
Tobacco +Arabid Rub +Arabid Raf1
co-expression with Raf1 increased foreign rubisco conten by >2 fold
BUT still lower than WT arabidopsis therefore ust be other chaperones
experiment by Salesse-Smith 2018
overexpressed rubisco subunits and overexpressed Raf1 genes
-increase rubisco in C4 maize by agrobacterium mediated transfer
small su only = no increase in rubisco
small su + Raf1 genes = more rubisco –> have incrased growth compared to WT!!!
increase in Rubisco in C4 does what
an increase from 12% to 14% soluble protein increased PS
oliver 2018
increase in Rubisco in C3 does what
no inrease in PS
no physiologicl enhancement
metabolic load in C3 plant already met
is C:O2 limiting not amount of rubisco ????
increase in Rubisco in algae does what
rubisco in algae is only 4% of soluble protein
increase PS rate
i dunno if it does or if this is twhat they want it to do
algae - biofuels, biomass, used for production of pharmaceuticals
how to engineer Rubisco more easily and why?
plant transformation take ages
speed it up by assmeblying in e. coli
study by Aigner did what?
engineer in e. coli 3 x plasmid 1 - RbcL (araB) 2- Cpn60/Cpn20 (T7) 3- Raf1/ RbcX, Bsd2 (T7)
diff promoter, T7 induced by sugars, incrased levels of chapeones before expressing rbcL gene
araB induced by arabinose in media
Results:
- PAGE-GEL results; confirmed requirements of each chapeone
- all 3 expression gave the highest rubisco expression in e.coli
- some worked even without Cpn20 - this enzyme is homologous to GroES, must be similar enough to substitute one another
what chapeones are general
Cpn60/20/10
what chapenones are rubisco specific
RbcX, Raf1, Raf2, raf3, Bsd2 etc
Study by Lin et al 2019 did what
modified study by Aigner 2017, to express tobacco genes and motiored chapeone + rubisco kinetics
altered small subunits used
the native to tobacco trichome small subunit has a higher catalytic rate but lower CO2 affinity compared to the other small subunits tested
tobacco rubisco successfully expressed and assembled in e.coli using PET and pCDF expression systems
