James P. Tam

Disulfides in peptides and proteins are essential for maintaining a properly folded structure. Their oxidative folding is invariably performed in an aqueous‐buffered solution. However, this process is often slow and can lead to misfolded products. Here, we report a novel concept and strategy that is bio‐inspired to mimic protein disulfide isomerase (PDI) by accelerating disulfide exchange rates many thousand‐fold. The proposed strategy termed organic oxidative folding is performed under organic solvents to yield correctly folded cysteine‐rich microproteins instantaneously without observable misfolded or dead‐end products. Compared to conventional aqueous oxidative folding strategies, enormously large rate accelerations up to 113,200‐fold were observed. The feasibility and generality of the organic oxidative folding strategy was successfully demonstrated on 15 cysteine‐rich microproteins of different hydrophobicity, lengths (14 to 58 residues), and numbers of disulfides (2 to 5 disulfides), producing the native products in a second and in high yield.

Coffee processing generates a huge amount of waste that contains many natural products. Here, we report the discovery of a panel of novel cell-penetrating and metal ion-binding microproteins designated coffeetide cC1a–c and cL1–6 from the husk of two popular coffee plants, Coffea canephora and Coffea liberica, respectively. Combining sequence determination and a database search, we show that the prototypic coffeetide cC1a is a 37-residue, eight-cysteine microprotein with a hevein-like cysteine motif, but without a chitin-binding domain. NMR determination of cC1a reveals a compact structure that confers its resistance to heat and proteolytic degradation. Disulfide mapping together with chemical synthesis reveals that cC1a has a ginsentide-like, and not a hevein-like, disulfide connectivity. In addition, transcriptomic analysis showed that the 98-residue micrcoproten-like coffeetide precursor contains a three-domain arrangement, like ginsentide precursors. Molecular modeling, together with experimental validation, revealed a Mg2+ and Fe3+ binding pocket at the N-terminus formed by three glutamic acids. Importantly, cC1a is amphipathic with a continuous stretch of 19 apolar amino acids, which enables its cell penetration to target intracellular proteins, despite being highly negatively charged. Our findings suggest that coffee by-products could provide a source of ginsentide-like bioactive peptides that have the potential to target intracellular proteins.