Protein Design

Question 1

What are some of the benefits in using directed evolution in protein design?

Answer 1

Proteins can be made which are used in making everything from biofuel to pharmaceuticals

i.e. antibodies using phage display for curing disease or washing powder enzymes to reduce washing temperatures thus using less energy

Question 2

What are some problems with photosynthesis?

Answer 2

Only 5 percent of the light energy captured is converted into glucose

Rubisco is very inefficient

As temperature increases (as with climate change) Rubisco will choose oxygenation over carboxylation

Phosphoglycerate (3-PGA), a product of Rubisco activities, can be toxic in large amounts to plants

Rubisco is a very promiscuous enzyme and will catalyse multiple reactions

As the concentration of carbon dioxide increases, the Kcat of Rubisco will decrease

Question 3

What is one way in which photosynthesis can be made more efficient?

Answer 3

Increasing chlorophyll levels to capture different wavelengths of light

Question 4

What are some benefits of using directed evolution over normal evolution?

Answer 4

Evolution can’t predict the future

1 mutation occurs at a time which is slow and inefficient

One phenotype may increase an organism’s fitness, but that may be less fit than the optimal for the conditions or fitness may decrease with one mutation before increasing with another

Question 5

Describe the directed evolution cycle

Answer 5

Some versions of DNA polymerase aren’t very accurate which can give to rises in sequence mutations and this is called random mutagenic PCR

In the 50’s this was done using radiation to damage the DNA and give rise to mutations

Random mutagenic PCR produced a gene library with lots of sequence mutations

This library is then expressed in E. coli or yeast

Activity assays are then conducted to see which mutation gave rise to the fittest and these desired variants are isolated and the genes they contained are also isolated

Question 6

What is gene shuffling and how is it conducted?

Answer 6

The generation of sequence libraries containing the information from a family of related genes

Target genes are digested using DNase I to produce fragmented DNA

PCR is then conducted without primers so the fragments anneals and then are extended

PCR with primers is then conducted to produce shuffled genes

Question 7

Describe how Rubisco can exemplify protein design by directed evolution (RDE screens)

Answer 7

Rubisco dependent E. coli screens (RDE) rely on the toxicity of RuBP, a foreign metabolite produced from the pentose phosphate pathway

RDE screens utilise a phosphoribulokinase (PRK)- neomycin phosphotransferase (NPTII) fusion where NPTII is attached to the C terminus of the PRK which renders kanamycin resistance to E. coli unless PRK-NPTII expression is transposon silenced

Successive evolution rounds using the plant-like Te-Rubisco from the cyanobacterium Thermosynechococcus elongatus BP1 identified two large subunit and six small subunit mutations that improved carboxylation rate, efficiency, and specificity

However, cyanobacterial Rubisco is not compatible with terrestrial plants due to the carboxysomes around Rubisco active sites

Question 8

What is the equation for the rate of nucleotide substitution?

Answer 8

r=K/2T

r= rate of nucleotide substitution

K= number of substitution between 2 sequences

T= time of divergence

Question 9

What are the 2 types of mutations?

Answer 9

Synonymous and non-synonymous

Question 10

What are synonymous mutations?

Answer 10

Where changing a nucleotide doesn’t change the amino acid

All synonymous mutations are neutral

Question 11

What are non-synonymous mutations?

Answer 11

Where changing a nucleotide changes the amino acid and can be advantageous, disadvantageous or neutral

Mutations that are advantageous undergo fixation faster than neutral or disadvantageous

Question 12

What is dN/dS?

Answer 12

dN/dS is the ratio of the number of nonsynonymous substitutions per non-synonymous site (pN) to the number of synonymous substitutions per synonymous site (pS)

Question 13

What happens if dN/dS is > 1 ?

Answer 13

This means positive selection and is advantageous

Question 14

What happens if dN/dS = 1 ?

Answer 14

Strictly neutral thus the gene is redundant

Question 15

What happens if dN/dS < 1 ?

Answer 15

This means negative selection thus the gene is disadvantageous

Question 16

What are some statistics about rates of positive selection?

Answer 16

In a study of 5641 mouse-rat gene comparisons of nonsynonymous/synonymous rates, the majority were <1, a few were =1 and very few were >1

Thus positive selection is very rare

Question 17

What is the relationship between positive selection and land plants v algae?

Answer 17

Positive selection is widespread in Rubiscos of land plants and algae but this is restricted to a small number of sites

Question 18

Why are Rubiscos in different plants different?

Answer 18

Rubisco adapts to different climates, leading to mutations and changes in amino acids

Rubiscos in the same plants but due to global distribution, they may be slightly different

Question 19

How can machine learning be applied to modifying Rubisco?

Answer 19

RbcL, which is a gene responsible for the production of Rubisco, when modified using amino acid replacements, can increase the Kcat of Rubisco in tobacco (Kapralov & Whitney, unpublished)

Rubisco kinetics can be predicted from RbcL sequence data using machine learning (Iqbal et al, 2022)

Protein structures of Rubiscos can be accurately predicted using AlphaFold (Jumper et al, 2021)

Question 20

Describe how Rubisco can exemplify protein expression

Answer 20

Heterologous protein expression and chaperones can be exemplified by Rubisco

Efficient in vivo folding of cellular and recombinant proteins requires molecular chaperones and foldases in the crowded cellular environment

Question 21

What type of evolution is the difference between green and red-type Rubiscos?

Answer 21

Convergent evolution- same shape and function but different genes

Question 22

Why can’t different Rubiscos simply be placed in crop plants to improve their substrate turnover?

Answer 22

Foreign Rubisco assembly has been found to decrease in tobacco plants with increasing phylogenetic distance (Kapralov & Whitney, unpublished) due to the different chaperones involved in the biogenesis and maintenane of Rubisco (Bracher et al, 2017)

However it was found that co-expression of foreign RAF1 (provides instructions for making a protein that is part of the RAS/MAPK pathway) and foreign Rubisco increases transgenic tobacco growth (Whitney et al, 2015), and over-expression of Rubisco subunits with RAF1 increases maize Rubisco content (Salesse-Smith et al, 2018)