Abstract
The reducing and capping sites along with their local structure impact photo properties of the red bovine serum albumin-capped Au nanocluster (BSA-AuNC), however, they are hard to identify. We developped a workflow and relevant techniques using mass spectrometry (MS) to identify the reducing and capping sites of BSA-AuNCs involved in their formation and fluorescence. Digestion without disulfide cleavages yielded an Au core fraction exhibiting red fluorescence and [AunSm] ion signals and a non-core fraction exhibiting neither of them. The core fraction was identified to mainly be comprised of peptides containing cysteine residues. The fluorescence and [AunSm] signals were quenched by tris(2-carboxyethyl)phosphine, confirming that disulfide groups were required for nanocluster stabilization and fluorescence. By MS sequencing, the disulfide pairs, C75–C91/C90–C101 in domain IA, C315–C360/C359–C368 in domain IIB, and C513–C558/C557–C566 in domain IIIB, were identified to be main capping sites of red AuNCs. Peptides containing oxidized cysteines (sulfinic or cysteic acid) were identified as reducing sites mainly in the non-core fraction, suggesting that disulfide cleavages by oxidization and conformational changes contributed to the subsequent growth of nanoclusters at nearby intact disulfide pairs. This is the first report on precise identification of the reducing and capping sites of BSA-AuNCs.
Highlights
Proteins have been implicated as the primary active biomolecules involved in biomineralization [1,2,3]
The occurrence of fluorescence resonance energy transfer (FRET) with Bovine serum albumin (BSA)-Au-NC was attributed to the interaction between tryptophan and AuNC as addressed in the literature [25,33] based on a significant decrease in intensity and fluorescence lifetime of tryptophan when both were excited at 295 nm [33]
Based on a workflow using several techniques relevant to protein mass spectrometry (MS), we identified key methionine (M445, M547) and cysteine (C34, C123, C264, C277, C460) residues of BSA responsible for Au+ reduction and the growth of AuNCs
Summary
Proteins have been implicated as the primary active biomolecules involved in biomineralization [1,2,3]. Protein scaffolds provide unique metal coordination environments that promote biomineralization processes [4,5]. The different shapes of gold nanoclusters were synthesized based on the template of different pH-dependent isoforms, but only the aged isoform (pH 12) yields large nanoclusters with red fluorescence [19,20]. Unlike a smaller close shell structure of nanoclusters (such as the internal, and potentially cascaded, energy transfers (FRET) between nanoclusters and Molecules 2019, 24, 1630; doi:10.3390/molecules24081630 www.mdpi.com/journal/molecules
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