L16 : Protein Folding on Ribosome Flashcards
What is co-translational folding on the ribosome?
Coupling of translation and folding so N-terminus end of NC begins to fold whilst C-terminal still in synthesis
Essential mechanism to ensure safe and efficient production and assembly of new proteins in their functional native states
How is protein folding on the ribosome different to in vitro?
Mostly understood through isolated polypeptides in vitro
Protein synthesis slower than protein folding
Occurs vectorially, 1 AA at a time from N-terminus
NC will begin trying to fold on co-translationally on ribosome (~20% human proteome assembles coT)
Intermediates will form to protect from aggregation and non-specific interactions
NCs will interact with chaperones co-translationally (TF, Hsp70)
Give an example for when co-translational folding is important for proteostasis
In vitro folding results in functionally inactive HRAS protein
When produced on ribosome, far more stable and higher activity
What were features of RNCs created to represent snapshots in biosynthesis and folding?
<1% total ribosome complex
Tethered to 2.5 MDa ribosome
Large sample requirements (several L)
Low molar conc (~15 um)
Limited sample lifetimes (12-24h at 298K)
Required ~100 samples per manuscript
What does cryo-EM of RNCs show
High resolution for ribosomal RNA and proteins
NC is highly dynamic and largely extended within exit tunnel due to limited dimensiosn
Folding only occurs significantly once NC has emerged from ribosome
Why can complexes only form outside of ribosomal tunnel?
Dimensions are limited (L=100A, W=10-20A) and can only fit ~30 AA when fully extended
Impossible for tertiary structure to form
How was NMR and truncated models used to mimic emerging NC?
NMR probes molecular dynamics to investigate disorder and order so ideal for considering transitions towards folded structure
Truncated models created for immunoglobulin domain (106 AAs) starting from N-terminus with 21 AAs left to add
As AAs added, spectra shows higher dispersion of peaks, representing transition from unfolded to fully folded sate
Can mimic coTF with isolated proteins
How were structures produced of proteins during folding?
Made a range of RNCs with increasing lengths of emerged NC
Can merge data from cryo-EM and NMR and combine computationally using molecular dynamics techniques.
How was site selective labelling of RNCs done for FLN5?
Introduced F label within NC in non-natural amino acids, simplifying the spectrum and allowing probing
Produces distinct NMR signal
Phenylalanine at position 655 replaced trifluoromethyl phenylalanine
What did 19F spectra for FLN5 show?
Showed that FLN5 is a 4-state folder when folding co-translationally on ribosome
Can observe 2 well populated intermediate states, stabilised by the ribosome
Conclusion: folding is different on the ribosome
Why do folding dynamics differ on the ribosome?
Major conformational expansion conferred by ribosome - U state of NC more expanded than isolated protein. (>30% increase in Rg)
Expansion partly caused by steric exclusion from tethering to ribosome, restricting conformational space available for folding
NC more expanded on ribosome, but also number of conformations it can adopt is limited - NC cannot fold all at once
U state is entropically destabilised on ribosome, promoting formation of folding intermediates
Why is it thought that U state is entropically destabilised on ribosome
More expanded chain traps more water mols on ribosome, decreasing entropy of solvation
Fewer conformations are sampled by U state on ribosome than off, decreasing conformational entropy
Entropic destabilisation makes U state less stable and may promote early folding events and formation of intermediates
Net increase in free energy of U state on ribosome by +9 kcal/mol
