Post-translational modifications Flashcards
Why is using mammalian cell lines as a model system challenging?
When would we use it?
Why is bacteria/e.coli better?
What is their drawback?
What about yeasts?
Need to maintain culture at right conditions
expensive medium
correct lab set up to grow cells consistently
to make complex mammalian proteins
Simpler proteins
Medium less expensive
Rapid cell growth
higher productivity
Simple hormones, industrial enzymes
not a lot of post trans modifications
Eukaryotic system - genetically tracked (like E. coli)
Easy grow- rapid on industrial scale
Has capacity to make more complex eukaryotic proteins
How is N-terminal methionine or formyl-methioning (bacteria, plasmids, mitochondria) removed in 2 steps?
What is the problem with methionine in eukaryotes & archaea?
What is the N-end rule pathway?
What are the destabilising residues in bacteria?
- Formyl H-C=O removed by deformylase
- Aminopeptidase removes methionine
Protein is inactive
Size of 2nd residue after methionine N-terminus is the stability determinant - so protein degradation is decided on whether the 2nd residue stabilises or destabilises the protein
R K M
What does the formation of intra/iner molecular disulphide bonds to do the proteins?
In what conditions does this happen?
What type of protein requires disulphide bonds?
What is an isopeptide bond?
When does it form?
GFP autocatalytic cyclisation of 3 adjacent residues lead to the chromophore (ser65, tyr66, gly67). What does the formation of the fluorophore rely on?
Helps stabilise & help fold into correct conformation
Oxidising environment
Antibodies
Covalent link between carboxyl & amino group between different side groups
Under initial folding & in hydrophobic conditions - isopeptide bond is stabilised by hydrogen bonds
Hydrophobic reaction & oxygen to form saturated rings
What is the processing of pre-pro insulin lead to?
What is required?
How does protein splicing also process pre-proteins?
What does this allow for?
What are some examples of post-translational covalent attachment to proteins?
What are some examples of post-translational modifications to amino acid residues?
Why are they required in bacteria?
Two polypeptides held together by disulphide bonds - formation of insulin
Enzymes expressed by E. coli
Remove inteins allowing new covalent peptide bond to form between N and C exteines
Design genes to produce mature proteins - without inteins
Suno, disulphide bonds & ubiquitin to lysine residues
Phosphorylation, glycosylation, acetylation etc
to make protein function- less extensive in eukaryotes
How do proteins assembled into quaternary structure?
What is the problem with rubisco?
Why is it more favourable to produce recombinant rubisco in e. coli?
What is the problem with using e. coli as a model system?
What are some examples of co-factors added to a protein?
What needs to be considered when expressing enzymes in a foreign host?
Protein subunits finally associate into final structure
Slow catalytic rate, can’t distinguish CO2/O2 well (fixes CO2 into organic form), requires assembly factors
Can express several other genes to assemble it into a functioning protein
Over expression leads to aggregation polypeptides into inclusion bodies
Haems, vitamins, metals etc
Consider co-factors required for the enzyme to be fully functioning & active
How is protein targeting in eukaryotes a PTM?
How do the Sec and Tat pathways act & what are their function?
What does the sec system do?
What does the Tat pathway do?
How does the unfolded peptide translocate across the membrane with the Sec system?
How does the Tat pathway export folded proteins?
How could you modify a peptide which has multiple targets?
Signal sequences determine protein location - add signal sequence for its activation
Operate in parallel to transport proteins across bacteria cytoplasmic membrane
Transports unfolded proteins co or post-translationally into membrane & fold in extracellular compartment
Exports folded protein so only post-translational
SRP, peptide signal sequence & ribosome, or unfolded peptide and SecA
Produce monomers that form a ring around the protein - ring interacts with membrane & allow protein translocate
Add multiple signal sequences
