Iminothioindoxyl Donors with Exceptionally High Cross Section for Protein Vibrational Energy Transfer.

Various protein functions are related to vibrational energy transfer (VET) as an important mechanism. The underlying transfer pathways can be experimentally followed by ultrafast Vis-pump/IR-probe spectroscopy with a donor-sensor pair of non-canonical amino acids (ncAAs) incorporated in a protein. However, so far only one donor ncAA, azulenylalanine (AzAla), exists, which suffers from a comparably low Vis extinction coefficient. Here, we introduce two novel donor ncAAs based on an iminothioindoxyl (ITI) chromophore. The dimethylamino-ITI (DMA-ITI) and julolidine-ITI (J-ITI) moieties overcome the limitation of AzAla with a 50 times higher Vis extinction coefficient. While ITI moieties are known for ultrafast photoswitching, DMA-ITI and J-ITI exclusively form a hot ground state on the sub-ps timescale instead, which is essential for their usage as vibrational energy donor. In VET measurements of donor-sensor dipeptides we investigate the performance of the new donors. We observe 20 times larger signals compared to the established AzAla donor, which opens unprecedented possibilities for the study of VET in proteins.

Versatile Vibrational Energy Sensors for Proteins.

Vibrational energy transfer (VET) is emerging as key mechanism for protein functions, possibly playing an important role for energy dissipation, allosteric regulation, and enzyme catalysis. A deep understanding of VET is required to elucidate its role in such processes. Ultrafast VIS-pump/IR-probe spectroscopy can detect pathways of VET in proteins. However, the requirement of having a VET donor and a VET sensor installed simultaneously limits the possible target proteins and sites; to increase their number we compare six IR labels regarding their utility as VET sensors. We compare these labels in terms of their FTIR, and VET signature in VET donor-sensor dipeptides in different solvents. Furthermore, we incorporated four of these labels in PDZ3 to assess their capabilities in more complex systems. Our results show that different IR labels can be used interchangeably, allowing for free choice of the right label depending on the system under investigation and the methods available.