Figure 1.
Effects of biotinylation on lysozyme.
(A) MALDI mass spectrometry of lysozyme incubated in the absence (upper) and the presence (lower) of 100-fold molar concentration of BioNSE. (B) Primary sequence of human lysozyme showing the sites where trypsin digestion has occurred in BioHuL (open grey arrows) and WTHuL (filled black arrows). (C) Secondary structure of BioHuL (dashed grey line) and WTHuL (solid black line) by far-UV CD. (D) Thermal denaturation curves of BioHuL (open grey circles) and WTHuL (filled black squares) monitored by near-UV CD. Mid-point Tm values are defined as the temperatures at which 50% of the population of protein molecules is unfolded.
Figure 2.
Mapping the location of modification by NMR spectroscopy.
(A) Overlaid HSQC NMR spectra at 700 MHz of BioHuL (red) and WTHuL (blue); the spectra were collected at pH 5.0 and 37°C (B) Chemical shift changes defined as [0.04(δ15NWT−δ15NBiotin)2+(δ1HWT−δ1HBiotin)2]1/2 in BioHuL with respect to WTHuL [68]. (C) Structural identification of the BioHuL protein colour-coded by Δδ value of each residue observed in (B) with the modified Lys33 labelled in red. Dark blue represents the lowest Δδ value, whilst red represents the highest. Residues whose chemical shifts are most perturbed by the modification are identified. The black line in the centre of the two images represents the axis of rotation by 180°.
Figure 3.
(A) In situ Thio-T binding fluorescence kinetics of BioHuL (grey) and WTHuL (black) incubated in the presence of 3 M urea in 0.1 M citrate buffer (pH 5.0) with constant stirring at 60°C. (B) TEM images of the endpoint samples of the aggregation reactions performed in the absence Thio-T. Images on the left and right show the fibrils formed by WTHuL and BioHuL, respectively; the scale bars represent 500 nm. (C) Dot blot assay of fibrils formed by WTHuL (upper row) and BioHuL (bottom row). Samples include the monomeric protein, the supernatant solutions after washing the fibril pellets (1st, 2nd, and 3rd) and the final washed fibrils. The appearance of colour indicates a positive interaction between biotin and streptavidin-AP. (D) In situ Thio-T binding fluorescence kinetics of WTHuL in the absence (solid line) and presence of 10% BioHuL fibril seeds (dashed line) incubated under similar conditions as (A). (Inset) TEM images of the WTHuL fibrils formed in the presence of BioHuL fibril seeds; the scale bar represents 500 nm. (E) Conformational stability of BioHuL (grey open circles) and WTHuL (black squares) fibrils. The stability of the fibrils was measured by depolymerisation experiments performed using GdnHCl at pH 5.0. Continuous lines represent the best fits to sigmoidal functions.
Figure 4.
dSTORM images of BioHuL fibrils.
(A)–(C) Super-resolution dSTORM images of different BioHuL fibrils (formed in vitro). (D) An overlay of a straight BioHuL fibril with its fluorescence sum image. (E) The cross-sections of the individual fluorescence sum and the super-resolved dSTORM image of the BioHuL fibril displayed in panel (D). The full-width half-maximum (FWHM) of the fluorescence intensity distribution of the unresolved sum image depicts a fibril diameter of 783 nm whereas the super-resolved image depicts a fibril diameter of 133±20 nm; the latter showing 6 times better resolution. (F) (left panel) DIC image of BioHuL present within SH-SY5Y mammalian cells after probing with streptavidin-Alexa647. (middle panel) Fluorescence sum image of a region within the SH-SY5Y cells (right panels) Super-resolution dSTORM images of BioHuL fibrils in the same region of the SH-SY5Y mammalian cells as the fluorescence sum image. All scale bars all represent 1 μm.