Allosteric information transfer in proteins has been linked to distinct vibrational energy transfer (VET) pathways in an umber of theoretical studies.E xperimental evidence for such pathways, however,i ss parse because site-selective injection of vibrational energy into aprotein, that is,localized heating,isrequired for their investigation. Here,wesolved this problem by the site-specific incorporation of the non-canonical amino acid b-(1-azulenyl)-l-alanine (AzAla) through genetic code expansion. As an exception to Kashasr ule,A zAla undergoes ultrafast internal conversion and heating after S 1 excitation while upon S 2 excitation, it serves as af luorescent label. We equipped PDZ3, ap rotein interaction domain of postsynaptic density protein 95, with this ultrafast heater at two distinct positions.W eindeed observed VET from the incorporation sites in the protein to ab ound 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 aw ide range of proteins. Dr.J .Jaric Present address:H ospira Zagreb d.o.o.,aPfizer company Prudnicka cesta 60, 10291 Prigorje Brdovecko (Croatia) [ + + ]T hese authors contributed equally to this work. Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

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 β-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.

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.

Eine Reihe theoretischer Studien setzt allosterischen Informationstransfer in Proteinen in Beziehung zu definierten Schwingungsenergietransfer(VET)‐Pfaden. Experimentelle Evidenz für diese Pfade ist rar, da für ihre Erforschung eine punktuelle Injektion von Schwingungsenergie in Proteine, d. h. eine lokale Erhitzung, benötigt wird. Um diese Experimente zu ermöglichen, haben wir den ortsspezifischen Einbau der nichtkanonischen Aminosäure β‐(1‐Azulenyl)‐l‐alanin (AzAla) mithilfe des erweiterten genetischen Codes etabliert. AzAla stellt eine Ausnahme der Kasha‐Regel dar: während die Aminosäure bei S1‐Anregung interne Konversion durchläuft und dadurch erhitzt, kann sie bei S2‐Anregung als Fluoreszenzmarker dienen. Wir haben PDZ3, eine Interaktionsdomäne des postsynaptischen Proteins PSD‐95, an verschiedenen Positionen mit diesem ultraschnellen Heizelement ausgestattet. Unter Verwendung von ultraschneller IR‐Spektroskopie konnten wir VET, ausgehend von AzAla innerhalb des Proteins, zu einem gebundenen Liganden auf einer Pikosekunden‐Zeitskala beobachten. Dieser Ansatz basierend auf dem genetisch codierten Einbau von AzAla ebnet den Weg zu detaillierten Studien von VET und seiner Funktion in einer Vielzahl von Proteinen.