Fig 2: NMR characterisation of the N-terminal fragment AAT-191.a (upper) 1D and (lower) 2D 1H,15N-SOFAST HMQC spectra of the AAT-191 fragment in (left) 0 M and (right) 8 M urea, recorded at 25 °C and at a 1H frequency of 950 MHz. b Selected AAT-191 resonances observed in increasing concentrations of urea. c (upper) A Kyte and Doolittle hydropathy plot for AAT-191 (lower) A plot depicting the urea concentrations at which non-proline residues are first observable, and includes a 3-point moving average to highlight the extent of compaction observed across the sequence. d Mapped onto the AAT structure is a representation of AAT-191 depicting the regions that progressively unfold in 8 M urea and at which stage NMR resonances first become observable (c).

Fig 3: Differentiation of hESCs into hepatoblasts in defined xeno-free conditions.a The relative hepatoblast gene (HNF4α and AFP) expression levels of cells differentiated with a previously reported protocol were determined by qPCR at days 6, 7, 8, 9, and 10. b Four protocols were used to induce PE to hepatoblasts. Trf (transferrin, 5 mg/mL); Vc-Mg (L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 50 µg/L); Ins (insulin, 10 µg/mL); SS (sodium selenite, 0.1 ng/mL); and BMP4 (10 ng/mL); bFGF (10 ng/mL). c The relative hepatoblast gene (HNF4α and AFP) expression of day 9 differentiated cells in basal medium (DMEM/F12, 1 × L-GlutaMAX, and 1 × NEAA) with different treatments (groups A, B, C, and D) were determined by qPCR. d The relative hepatocyte gene (ALB, AAT, and CK18) expression of day 19 differentiated cells in basal medium (DMEM/F12, 1 × L-GlutaMAX, and 1 × NEAA) with different treatments (groups A, B, C, and D) were determined by qPCR. e Immunofluorescence analysis of HNF4α and AFP expression in group A-induced differentiated cells on day 9. f The expression levels of HNF4α and AFP in group A-induced differentiated cells were determined by flow cytometry on day 9. Isotype control antibodies were used as controls. *p < 0.05, **p < 0.01; data are represented as the mean ± SD. Scale bar, 100 µm.

Fig 4: Wild-type and Z AAT ribosome-nascent chain complexes (RNC) can adopt co-translational structure.a A schematic of the proteinase K limited proteolysis experiment (upper). Limited proteolysis of wild-type and Z purified RNCs (orange arrowhead), and purified, released wild-type AAT (blue arrowhead) monitored by an anti-His western blot (lower). Highlighted are two N-terminal proteolytic products released from the nascent chain segment emerged from the ribosome: 42 kDa (grey arrowhead) and 23 kDa (green arrowhead). b Schematic of an AAT RNC highlighting the N-terminal fragments observed in a (left). (right) Structure of AAT and schematic representation of (released) AAT showing the boundaries of the N-terminal (NTD, green) and C-terminal (CTD, grey) sub-domains, and C-terminal residues 360–394 which are occluded in the ribosomal tunnel within the RNC (see left). The segment of structure shown in black is the 35 amino acid stretch that is occluded in the tunnel when full-length AAT is bound to the ribosome. c Proteinase K limited proteolysis measured over time for wild-type and Z RNCs, highlighting the measurement of the intact tRNA-bound nascent chain (NC) over time using 35S-methionine detection and partially-denaturing PAGE (n = 3 biological repeats). See also Supplementary Fig. 2. d Densitometric analysis of the proteinase K time-course from c with exponential fits. Data are presented as mean values +/− SEM.

Fig 5: Monitoring the folding and misfolding of alpha-1-antitrypsin (AAT) during biosynthesis in rabbit reticulocyte lysate (RRL) using 35S methionine radiolabelling.a (upper) AAT structure (PDB ID: 1QLP) with landmarks highlighted: A-sheet (red), B-sheet (green), C-sheet (yellow), reactive centre loop (RCL, purple), and site of the Z mutation (E342K). (lower) Schematic of the full-length AAT DNA construct and highlighting the relative position of the Z (E342K) mutation. b Scheme of released AAT biosynthesis and folding in RRL. c Production of wild-type AAT nascent chains as followed by denaturing PAGE. Highlighted are the full-length species (arrow), and high molecular weight species / polymerisation (i.e., a smear) as a dashed line. d Folding of wild-type AAT nascent chains as monitored by native PAGE (using the same samples as in c). Highlighted is the biologically-active, monomeric species (arrow) and polymer (dashed line). e Production of Z nascent chains as followed by denaturing PAGE. Highlighted are the full-length species (arrow), and high molecular weight species / polymerisation (i.e., a smear) as a dashed line. f Folding of Z AAT nascent chains as monitored by native PAGE (using the same samples as in e). Unlike wild-type in c, Z nascent chains cannot fold efficiently (solid line) and form polymers (dashed line). g Biosynthesis and native folding of wild-type AAT nascent chains (analysis from c, d; n = 4). h Biosynthesis of wild-type and Z AAT nascent chains over 60 min. Data have been normalised to the reaction end-point to enable a comparative analysis. All samples were flash-frozen and treated with RNase A prior to analysis. (n = 5 biological repeats). Data are presented as mean values +/− SEM.