Fig. 6

The W344R mutation disrupts CRD folding during translation. a Fluorescent image of an SDS-PAGE gel of unboiled bacterial extracts of WT or W344R biosensors expressed at 37 °C. Proteins were co-expressed (right columns) or not (left columns) with CaM. Soluble (supernatant; SN) and insoluble (pellet; P) protein fractions were separated, and loaded as indicated. b Fluorescence intensity of the supernatant band of SDS-PAGE gels (n = 3). c Emission spectra of the soluble fraction of WT (black lines) and W344R (grey lines) proteins expressed alone (solid lines) or co-expressed with CaM (dashed lines). d FRET efficiency values from spectra as in C (WT n = 16, WT + CaM n = 21, W344R n = 12, W344R + CaM n = 27). e Top: Schematic representation of the constructs used for in vivo co-translation folding monitoring. The CRD was cloned upstream of the SecM arresting peptide (AP) sequence with tethers of increasing length, ranging from 18 to 50 amino acids from the C-terminal conserved Pro of the SecM AP where translational stalling takes place. mTFP1 and mcpVenus were fused to the N- and C-terminus, respectively. Folding events of the protein domains inside or outside the ribosomal tunnel alleviate SecM stalling and leads to an increase in the ratio of peak emission mcpVenus/mTPF1. Bottom: Co-translational folding profiles of the indicated AB CRD constructs, expressed with and without CaM. Number of experiments is indicated in brackets