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.

Through bonds or contacts? Mapping protein vibrational energy transfer using non-canonical amino acids.

Vibrational energy transfer (VET) is essential for protein function. It is responsible for efficient energy dissipation in reaction sites, and has been linked to pathways of allosteric communication. While it is understood that VET occurs via backbone as well as via non-covalent contacts, little is known about the competition of these two transport channels, which determines the VET pathways. To tackle this problem, we equipped the ß-hairpin fold of a tryptophan zipper with pairs of non-canonical amino acids, one serving as a VET injector and one as a VET sensor in a femtosecond pump probe experiment. Accompanying extensive non-equilibrium molecular dynamics simulations combined with a master equation analysis unravel the VET pathways. Our joint experimental/computational endeavor reveals the efficiency of backbone vs. contact transport, showing that even if cutting short backbone stretches of only 3 to 4 amino acids in a protein, hydrogen bonds are the dominant VET pathway.

Site-Resolved Observation of Vibrational Energy Transfer Using a Genetically Encoded Ultrafast Heater.

Allosteric information transfer in proteins has been linked to distinct vibrational energy transfer (VET) pathways in a number of theoretical studies. Experimental evidence for such pathways, however, is sparse because site-selective injection of vibrational energy into a protein, that is, localized heating, is required for their investigation. Here, we solved this problem by the site-specific incorporation of the non-canonical amino acid ß-(1-azulenyl)-l-alanine (AzAla) through genetic code expansion. As an exception to Kasha's rule, AzAla undergoes ultrafast internal conversion and heating after S1 excitation while upon S2 excitation, it serves as a fluorescent label. We equipped PDZ3, a protein interaction domain of postsynaptic density protein 95, with this ultrafast heater at two distinct positions. We indeed observed VET from the incorporation sites in the protein to a bound peptide ligand on the picosecond timescale by ultrafast IR spectroscopy. This approach based on genetically encoded AzAla paves the way for detailed studies of VET and its role in a wide range of proteins.