During the last decade, a great variety of ligand protected gold nanoclusters (AuNCs) have been synthesized, and their broad applications have been intensively reported. Although the spectroscopic properties of AuNCs have been comprehensively explored, the mechanism of the significant Stokes shift (> 200 nm) and the specific role played by surface ligands have not been clearly explained yet. In this study, a series of fluorescent AuNCs with huge Stokes shift (up to 530 nm) were successfully prepared by employing the rationally designed tri-peptides as the protecting ligands, and their spectroscopic properties were systematically investigated. The detailed measurements on the example product, YCY-AuNCs (Tyr-Cys-Tyr liganded AuNCs), showed that the energy absorbed by the tyrosine (~ 250 nm) can be effectively transferred through the ligand-mediated two-step Förster resonance energy transfer (FRET) process and released as fluorescence emission in the near-infrared fluorescence (NIR) range (~ 780 nm), which resulted in the significant apparent Stokes shift. The YCY ligands play a critical role by offering the tyrosine groups (donor of the first FRET pair), generating the dityrosine-like structure on the AuNCs surface (acceptor of the first FRET pair and donor of the second FRET pair), and protecting the cores (acceptor of the second acceptor). The additional ligand exchange experiments and the investigation on the other AuNCs further demonstrated that the sufficient high density of the aromatic groups is also essential to mediate the two-step FRET and achieve the remarkable Stokes shift. We believe that the aromatic ligand-mediated FRET mechanism not only offers a new theoretical explanation for the huge Stokes shift exhibited in AuNCs, but also provides a general strategy for the construction of new materials with large Stokes shift.

A novel peptidomimetic-liganded gold nanocluster (CDp-AuNC) is proposed for the synergistic suppression of tumor growth. Taking advantages of the multi-capabilities offered by the surface ligands, including iron chelation, glutathione peroxidases-1 (GPx-1) binding, and tumor cells recognition, CDp-AuNCs are able to function as the nanocarriers to deliver iron in a controlled manner for the ferroptosis therapy and as the inhibitors for GPx-1 to induce the apoptosis of tumor cells. The Fe²⁺@CDp-AuNC nanocomplexes are fabricated through a facile self-assembly method. The experimental data verify that the nanocomplexes are internalized specifically by tumor cells with high efficiency. The acidic microenvironment in endosomes triggers the collapse of the nanocomplexes and thereby releases Fe²⁺ to induce ferroptosis and CDp-AuNCs to inhibit the enzyme activity of GPx-1. Benefiting from the H₂O₂-depleted pathway inhibition and ferroptosis acceleration, the intracellular reactive oxygen species (ROS) level could be enhanced significantly. As a consequence, the apoptosis/ferroptosis of 4T1 cells as well as the tumor elimination in vivo are observed after treatment with the Fe²⁺@CDp-AuNC nanocomplexes at a relatively low dose. The facile iron loading method, simple construction procedure, and outstanding tumor suppression performance, provide CDp-AuNCs great application promise. More importantly, the strategy of peptidomimetic ligands design provides a transferable approach to building multifunctional nanomaterials.

Our understanding of molecular chaperone function in membrane protein biogenesis lags far behind that in soluble protein biogenesis. Through a combined approach including isothermal titration calorimetry, UV–Vis spectroscopy, and fluorescence spectroscopy, the behavior of ATP-dependent chaperonin GroEL–GroES, a paradigmatic chaperone of soluble protein folding, was investigated in the refolding of membrane protein bacteriorhodopsin (BR) and its membrane insertion. We found that BR bound asymmetrically to the double-ring GroEL, with a much higher affinity when it was partially denatured. GroEL alone showed a clear influence on BR refolding, but the presence of ATP was necessary to significantly enhance both the rate and yield of the GroEL-mediated folding, in contrast to the adverse effect of GroES on the folding yield. However, synergy between ATP and GroES was shown to be required not only for releasing high-affinity BR species from GroEL, but also for unfolding and rescuing the misfolded conformers complexed to GroEL. This is consistent with the observation that maximum rate enhancement of BR refolding or assembly with the prepared inverted membrane vesicles was achieved when the complete chaperonin system was used. Our results support the iterative unfolding mechanism of GroEL activity previously proposed for soluble proteins, whereby GroEL might perform repeated unfolding and release of BR, thus offering additional opportunities for timely folding or membrane integration. This work provides important information on the convergence of folding of membrane and soluble proteins in light of folding pathways and the role of molecular chaperones.