Revisiting the conformational state of albumin conjugated to gold nanoclusters: A self-assembly pathway to giant superstructures unraveled

Abstract

Bovine serum albumin (BSA) is often employed as a proteinaceous component for synthesis of luminescent protein-stabilized gold nanoclusters (AuNC): intriguing systems with many potential applications. Typically, the formation of BSA-AuNC conjugate occurs under strongly alkaline conditions. Due to the sheer complexity of intertwined chemical and structural transitions taking place upon BSA-AuNC formation, the state of albumin enveloping AuNCs remains poorly characterized. Here, we study the conformational properties of BSA bound to AuNCs using an array of biophysical tools including vibrational spectroscopy, circular dichroism, fluorescence spectroscopy and trypsin digestion. The alkaline conditions of BSA-AuNC self-assembly appear to be primary responsible for the profound irreversible disruption of tertiary contacts, partial unfolding of native α-helices, hydrolysis of disulfide bonds and the protein becoming vulnerable to trypsin digestion. Further unfolding of BSA-AuNC by guanidinium hydrochloride (GdnHCl) is fully reversible equally in terms of albumin’s secondary structure and conjugate’s luminescent properties. This suggests that binding to AuNCs traps the albumin molecule in a state that is both partly disordered and refractory to irreversible misfolding. Indeed, when BSA-AuNC is subjected to conditions favoring self-association of BSA into amyloid-like fibrils, the buildup of non-native β-sheet conformation is less pronounced than in a control experiment with unmodified BSA. Unexpectedly, BSA-AuNC reveals a tendency to self-assemble into giant twisted superstructures of micrometer lengths detectable with transmission electron microscopy (TEM), a property absent in unmodified BSA. The process is accompanied by ordering of bound AuNCs into elongated streaks and simultaneous decrease in fluorescence intensity. The newly discovered self-association pathway appears to be specifically accessible to protein molecules with a certain restriction on structural dynamics which in the case of BSA-AuNC arises from binding to metal nanoclusters. Our results have been discussed in the context of mechanisms of protein misfolding and applications of BSA-AuNC.

Enzymatic digestion of luminescent albumin-stabilized gold nanoclusters under anaerobic conditions: clues to the quenching mechanism

“…Next, SDS-PAGE electrophoresis was used to assess activity of proteinase K in the presence of DS. It was reported earlier that the synthesis of BSA-AuNC is accompanied by agglomeration of the protein into larger oligomers which remain nevertheless more susceptible to enzymatic proteolysis than native BSA 39 . As shown in the inset of Fig.…”

“…2D) is indicative of the dominant α-helical conformation even though its content in the conjugate is reduced relative to the native protein. 39 The fact that, unlike in native BSA, the CD intensity at 208 nm is roughly by one third higher than at 222 nm reflects the partial unfolding and destabilization of the helical fold taking place upon synthesis of the conjugate. The two superimposed spectra correspond to BSA-AuNC digested to 1 and 24 hours with proteinase K. Even the shorter digestion is sufficient to significantly disrupt the residual helical structure which is reflected both by exacerbated decay of the 222 nm component and the overall decrease of CD signal.…”

“…This approach has allowed us to study the impact of enzymatic digestion of protein envelope on BSA-AuNC luminescence under strictly anaerobic conditions and therefore draw more unambiguous conclusions regarding the oxygen's role in the quenching. The harsh conditions of BSA-AuNC synthesis render albumin molecules partly disordered 39 . Still, the surface of the native BSA molecule is often used to map residues which are more likely to become involved in the nesting of AuNCs.…”

The emergence of mass spectrometry for characterizing nanomaterials. Atomically precise nanoclusters and beyond

“…146 It is likely that ESI ionization efficiency is dramatically lowered due to the presence of counter ions from the synthesis procedure. 147 Following the first MALDI-MS spectrum reported by Xie and coworkers for BSA-templated gold nanoclusters, 148 efforts have been made, mainly by Pradeep's group, to use MALDI-MS to characterize protein-templated metal nanoclusters. 149 MALDI matrices are crystal-forming organic molecules used to enhance the vaporization of poorly-volatile analytes, and their ionization through charge transfer capability.…”

Potassium-Induced Emission Enhancement of Bovine Serum Albumin-Stabilized Red-Emitting Au Nanoclusters: Mechanism and Application to Blood Plasma

“…According to the EXAFS spectra analysis, luminescence intensity of glutathione-stabilized AuNCs was proportional to the degree of Au(I)-Au(I) bonds [36]. Recent transmission electron miscopy (TEM) images of BSA-rAuNCs, after a prolonged incubation, show the presence of long chains with triangularshaped side chains [41]. It is likely that the backbone chains in the images correspond to Au(I)-Au(I) linear chains and the side chains are Au(I)-(SR) residues.…”