Lecture 16 and 17 - Co-translational folding Flashcards
What is co-translational folding on the ribosome?
The essential mechanism by which cells ensure the safe and
efficient production and assembly of new proteins in their
functional native states.
What evidence is there to suggest that co-translational folding is important for proteostatis?
In vitro refolding of the HRAS protein resulted in the formation of a functionally inactive protein.
What are the features of Ribosome Nascent Chain complexes (RNCs) that were created?
<1% of total ribosome complex
Tethered to 2.5 Mda ribosome
Large sample requirements
(several L of culture /sample)
Low molar concentrations
~ 15 µM (= 37.5 mg/ml)
Limited sample lifetimes
12-24h at 298 K
~100 samples per manuscript
What do Cryo EMs of the RNCs show?
The nascent chain is extended
within the exit tunnel and is
dynamic.
Folding only occurs
significantly once the nascent
polypeptide has emerged from the
ribosome.
CryoEM of RNCs shows excellent
resolution for the ribosomal RNA
(rRNA) and proteins
The nascent chain is dynamic even
within the tunnel and the electron
density is weak.
Beyond the tunnel no CryoEM electron density corresponding to the emerged
chain is not seen as it is too dynamic
Why can complex structures form only outside the ribosome?
The dimensions of the ribosomal exit tunnel are a width of 20A, and length of 100A.
The length of the tunnel can fit around 30 amino acids.
The tunnel width makes it impossible for complex structure formation inside the tunnel.
How were truncation models used to mimic an emerging polypeptide chain?
We start with the N-terminal portion of the protein with 21 amino acids left to synthesise.
As amino acids are added, NMR spectra shows us a transition from the unfolded state to fully folded state.
At 9,8,6,4,2 amino acids left, we see a mix of folded and unfolded proteins.
What has NMR contributed to co-translational folding?
NMR relies on dynamics, so the more dynamic a molecule, the more intense the spectrum.
The 1H-15N spectrum shows one point for every amino acid. (Except proline)
Poor X-axis dispersion highlights unfolded states
High X-axis dispersion shows folded states.
How was a polypeptide chain studied coming off a ribosome?
By making a range of RNCs of
increasing lengths of emerged
nascent chain.
A 1H-13C fingerprint
shows the characteristics
of a well folded nascent chain.
We can go from this spectrum
to produce the first structure of
a nascent polypeptide ON its
parent ribosome
What did experiments with RNCs of different lengths show about the folding states of FLN5?
19F spectra can follow the folding process from completely unfolded (U) to the completely folded (F) state.
The folding process shows that it is NOT 2 state but rather there are unique co-translational intermediate states.
It is 4 state.
FOLDING is DIFFERENT ON THE RIBOSOME
Why is protein folding on the ribosome different from isolation?
the U states of the nascent chain is more expanded than the corresponding
isolated protein.
Expansion (partly) caused by steric exclusion from tethering to the ribosome.
The unfolded state is entropically destabilised on the ribosome,
The destabilisation of the unfolded state promotes the formation of folding intermediates.
Why is the The unfolded state is entropically destabilised on the ribosome?
There is a net increase in free energy of the unfolded state on the ribosome by +9 kcal/mol.
The more expanded chain on the ribosome traps more water molecules – this is entropically destabilizing.
Also the conformational entropy is also lower on the ribosome – fewer conformations are sampled by the unfolded state on the ribosome
compared to off the ribosome.
