Amir Bitran

@amirbitran.bsky.social

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Jane Coffin Childs fellow, Marqusee/ Bustamante Labs, UC Berkeley Prev. PhD Shakhnovich Lab, Harvard Protein folder, classical composer http://amirbitran.com

Nascent chain folding depends on kinetic competition between translation and folding rates. For details, see discussion in attached manuscript
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
(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.
Schematic of aTS folding. Solid colors refer to peptides with >10% natively protected population.
(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.
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).