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Amir Bitran @amirbitran.bsky.social · 05/07/2026

In summary, the ribosome uses diverse strategies to regulate protein folding, dependent not just on folding thermodynamics, but also kinetics + coupling to elongation. In the future, can extend this to study how co-translational folding links to evolutionary fitness and misfolding disease. (11/n)

Nascent chain folding depends on kinetic competition between translation and folding rates.
For details, see discussion in attached manuscript

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Amir Bitran @amirbitran.bsky.social · 05/07/2026

Notably, this minimal co-translational folding isn’t because HaloTag CAN’T fold on the ribosome, but rather it doesn’t have time to! When we artificially stall translation and give the nascent chain time to reach equilibrium, it actually populates an intermediate similar to that in refolding! (9/n)

$Fitted fractional populations associated with natively protected mode for indicated peptides from each RNC construct (opaque markers), and intermediate mode for peptide 37-66 (empty markers) (F) Native protected fraction vs time for the same peptides as in E but now as a function of time during active translation elongation$

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Amir Bitran @amirbitran.bsky.social · 05/07/2026

But something wildly different happens for HaloTag, which attains higher folding yield when translated vs refolded from denaturant. Turns out HT hardly folds on the ribosome, showing only weak HDX protection But once released, it folds through a different intermediate than when refolded! (7/n)

$(F) Schematic of HaloTag co-and-post translational folding with solid colors highlighting structures spanned by at least one peptide showing at least 50% natively protected fraction at the indicated times, striped colors indicating significant burst phase protection, white indicating no protection, and gray indicating no peptide coverage. (G) Same as (F) now depicting Halo Tag refolding from 7.5M urea.$

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Amir Bitran @amirbitran.bsky.social · 05/07/2026

We see something similar in a TIM barrel, aTS, where an N-terminal region roughly corresponding to a half barrel folds on the ribosome. Co-translational folding of TIM barrels has long been predicted, and we can now see it! Turns out this specific protein also folds similarly off the ribosome (6/n)

Schematic of aTS folding. Solid colors refer to peptides with >10% natively protected population.

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Amir Bitran @amirbitran.bsky.social · 05/07/2026

We can watch nascent proteins fold piece-by-piece! Here is some data for E. coli CMPK. Each line in panel C reflects folded fraction vs time for a specific stretch of residues during + after synthesis. We see a specific subdomain folds during synthesis, while the protein core folds after (5/n).

$(A) Structure of E. coli CMPK (PDB ID 2CMK) with colored HDX-MS reporter peptides, three of which are labeled.(C) Fractional population associated with natively protected mode vs. elongation time for indicated peptides. (D) Schematic of CMPK co-translational folding. Structures spanned by peptides showing >40% natively protected population at the indicated times are colored solid, provided the native mode is ≤60% deuterated, while peptides showing ≥60% fractional population in a partially protected mode (<80% of maximal deuteration, see Materials and Methods) are shown in dashed colors. Peptides satisfying neither condition are shown in white, and gray denotes structures lacking peptide coverage.$

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Amir Bitran @amirbitran.bsky.social · 05/07/2026

To solve this, I developed an approach based on hydrogen deuterium exchange mass spectrometry (HDX-MS) to detect the high-resolution, time-resolved folding of nascent proteins in a synchronized in vitro translation reaction! This entails enriching the “needle” out of the haystack as shown: (4/n)

Summary of experimental design from Fig. 2B. Synchronized translation is achieved by pre-initiating ribosomes on mRNA (step 1) followed by addition of complete amino acid mix and pre-charged tRNA to begin elongation (step 2) coupled to nascent chain biotinylation. At various elongation times, an aliquot is simultaneously pulsed with deuterium to label exposed amides and bound to streptavidin beads (step 3). The HDX reaction is quenched via acidification, which also dissociates ribosomes while nascent chains (NCs) remain bound to beads. (step 4). NCs are digested into peptides via addition of pepsin to the beads (step 5), then analyzed for deuteration via liquid-chromatography mass spectrometry (step 6).

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Amir Bitran @amirbitran.bsky.social · 28/12/2025

Thrilled to be giving a talk at the University of Chile tomorrow on my research (in spanish/spanglish)! Feel free to scan the QR code below, or contact me for a link if you're interested in joining!

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Amir Bitran @amirbitran.bsky.social · 26/11/2025

Really excited to be giving a seminar next week at @purduechemistry.bsky.social

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Amir Bitran @amirbitran.bsky.social · 08/03/2025

At the Stand up for science march, San Francisco

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