Fig 1.
(A) Luminescence emission spectrum of BSA-AuNC (λexc. = 365 nm) overlaid on unstained TEM images of BSA-AuNC; AuNCs of diameter below 1 nm are marked with red circles whereas larger non-fluorescent AuNPs are in black circles. Photographs of BSA-AuNC liquid sample illuminated with 365 nm UV light (top), and in daylight (bottom) are on the right. (B) Far-UV CD spectra of native BSA, BSA-Alk, and BSA-AuNC collected at the same protein concentration and pH 7; inset shows the corresponding near-UV CD spectra. (C) Raman spectra of native BSA and BSA-Alk (laser line 780 nm).
Fig 2.
SDS-PAGE analysis of susceptibility to digestion by trypsin of BSA, BSA-Alk, and BSA-AuNC under non-reducing conditions.
A portion of each sample corresponding to 7.5 μg of BSA mass was loaded onto each lane.
Fig 3.
Conformational stability of BSA-AuNC complex.
Titration of BSA-AuNC, native BSA, and BSA-Alk with GdnHCl at pH 7 and 25 oC monitored with far-UV CD. In the bottom right panel, quantitative plots correspond to GdnHCl-dependences of CD signals at 225 nm. Plasticity of secondary structures decreases in the order: BSA-AuNC > BSA-Alk > native BSA. The corresponding low GdnHCl concentration range is marked with the shadowed area.
Fig 4.
Reversibility of GdnHCl-induced unfolding of BSA-AuNC probed by luminescence of AuNC excited at 365 nm (A) and far-UV CD (B). The 0.6 M* label corresponds to initially 10-times more concentrated BSA-AuNC samples subjected to 6 M GdnHCl and subsequently ‘refolded’ by 10-times dilution with water; sharp peak marked with an asterisk corresponds to scattered 2*λexc light. The region of CD spectra collected in the presence of 6M GdnHCl corresponding to shortest wavelengths is perturbed due to strong UV absorption by the denaturant.
Fig 5.
(A) ATR-FTIR spectra (left: original absorption, right: corresponding second derivative) of aggregates: {BSA-AuNC}, {BSA-Alk}, and {BSA} obtained through 96h-long incubation of corresponding soluble precursors at pH 7.4, 75 oC compared with the spectra of native BSA; (B) The corresponding far-UV CD spectra and fluorescence emission spectra after staining with Thioflavin T (inset), the assignment of colors is the same as in panel (A).
Fig 6.
TEM images of BSA-AuNC complexes before (A), and after aggregation (B-F). The singly-dispersed AuNCs (A) associate into elongated entities upon prolonged incubation of BSA-AuNC complexes at pH 7.4, 75 oC–e.g. the encircled groups in panel (C). The progressing aggregation is accompanied by a gradual decrease in fluorescence intensity (excited at 365 nm), as reflected by the emission spectra overlaid in panel (B), the intense spike marked with (*) corresponds to scattered excitation beam. {BSA-AuNC} form regular superstructures visible on larger length scales (D-F). In the magnified image shown in inset of panel (D), the dark streaks are revealed to consist of elongated groups of AuNCs. The self-association behavior of {BSA-AuNC} is not observed for aggregates of BSA alone (inset image in panel (E) obtained after negative staining with uranyl acetate).
Fig 7.
Specimen of early diffuse {BSA-AuNC} aggregates co-existing with mature superstructures, as observed by TEM.
The mold-like entities reveal AuNCs being already pre-associated into stretched elongated groups before the superstructural self-assembly is completed.
Fig 8.
BSA: a 3D structure of native protein (A), PDB entry 3V03 [51], and an MD-generated snapshot of alkaline-disordered state (B). Various colors mark regions involved in AuNC-binding according to [22]. Regions with high propensity to aggregate mapped according to Tango algorithm [49–50]. Full amino acid sequence of BSA with likewise highlighted AuNC-binding and aggregation-prone regions (C). For MD-derived snapshot CHARMM package [52] and implicit solvent FACTS22 method [53] were used. Disordered conformations were obtained by 10-ns-long thermal unfolding of BSA with all disulfide bonds reduced (550 K, pH 12). Visualization was done with VMD program [54].