An ultrafast phototrigger of the Trp5CN-Trp motif in a ß-hairpin peptide.

Phototriggers are useful molecular tools to initiate reactions in enzymes by light for the purpose of photoenzymatic design and mechanistic investigations. Here, we incorporated the non-natural amino acid 5-cyanotryptophan (W5CN) in a polypeptide scaffold and resolved the photochemical reaction of the W5CN-W motif using femtosecond transient UV/Vis and mid-IR spectroscopy. We identified a marker band of ∼2037 cm-1 from the CN stretch of the electron transfer intermediate W5CN·- in the transient IR measurement and found UV/Vis spectroscopic evidence for the W·+ radical at 580 nm. Through kinetic analysis, we characterized that the charge separation between the excited W5CN and W occurs in 253 ps, with a charge-recombination lifetime of 862 ps. Our study highlights the potential use of the W5CN-W pair as an ultrafast phototrigger to initiate reactions in enzymes that are not light-sensitive, making downstream reactions accessible to femtosecond spectroscopic detection.

Probing Intermolecular Interactions of Amyloidogenic Fragments of SOD1 by Site-Specific Tryptophan and Its Noncanonical Derivative.

Transient amyloid intermediates are likely to be cytotoxic and play an essential role in amyloid-associated neurodegenerative diseases. Characterization of their structural and dynamic evolution is the key to elucidating the molecular mechanism of amyloid formation. Here, combining circular dichroism (CD), exciton couplet theory, and Fourier transform infrared spectroscopy with site-specific tryptophan (Trp) and its noncanonical derivative 5-cyano-tryptochan (Trp5CN), we developed a method to monitor strand-to-strand tertiary and sheet-to-sheet quaternary interactions in the aggregation cascades of an amyloidogenic fragment from protein SOD128-38 (with the sequence KVKVWGSIKGL). We found that the exciton couplet generated from the Bb band of Trp can be used as a probe for side chain interactions. Its sensitivity can be further improved by four times with the incorporation of Trp5CN. We further observed a red-shift of ∼2 cm-1 and a broadening of ∼2 cm-1 in the IR band generated from the CN stretch during the aggregation, which we attributed to the transition from a corkscrew-like structure to a cross-linked intermediate phase. We show here that the integration of optical methods with unique aromatic side chain-related probes is able to elucidate amyloid intermolecular interactions and even capture elusive transient intermediates on and off the amyloid assembling pathway.