Fig. 3: The AesN precursor 1P exhibits two folding states as revealed by SEC. | Nature Communications

Fig. 3: The AesN precursor 1P exhibits two folding states as revealed by SEC.

From: A cysteine-less and ultra-fast split intein rationally engineered from being aggregation-prone to highly efficient in protein trans-splicing

Fig. 3: The AesN precursor 1P exhibits two folding states as revealed by SEC.The alternative text for this image may have been generated using AI.

a SEC analysis of 1 P (left panel). This data and a calibration curve generated with globular protein standards were used to determine the Stokes radius (middle panel) and the apparent molecular weight of the aggregated (A) and monomeric (M) species. Because the aggregated species eluted shortly before dextran as the void volume marker, we could only estimate its hydrodynamic radius to be >270 Å. b Analysis of aggregated and monomeric fractions on a Coomassie-stained SDS-PAGE gel. c Analysis of PTS reactions using the aggregated and monomeric species of 1P (10 µM) with three-fold excess of 3P (30 µM) at 37 °C. Shown are Coomassie-stained SDS-PAGE gels. d Time-courses of two PTS reactions using monomeric fractions of the indicated AesN precursors 1P and 4P with a three-fold excess of 3P (see also Supplementary Fig. 4). e Analysis of concentration-dependent re-aggregation of 1P over time. The left panel shows representative analytical SEC profiles recorded at indicated time points following the isolation of its monomeric fraction adjusted to 10 µM. The right panel shows the plot of the monomeric fraction. Note that only the second, exponential phase was used for fitting. The sigmoidal appearance of the curve at early time points is expected as a typical feature of amyloid aggregate formation due to a slow nucleation (lag phase) before aggregation proceeds as a concentration-dependent polymerization reaction (exponential phase) until reaching saturation (equilibrium phase)46. f Analysis of concentration-dependent resolution of 1P aggregate. The left panel shows representative analytical SEC profiles recorded at indicated time points after dilution of an aggregate fraction from 45 µM to 10 µM. The right panel shows the plotted aggregate resolution to the monomeric species from starting concentrations 135 µM and 45 µM, each fitted using a one-phase exponential equation. For (a) (middle and right inlets), n = 3 technical replicates. Data are presented as mean. Source data are provided as a Source Data file.

Back to article page