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.

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.

In the early stages of SARS-CoV-2 infection, non-structural protein 1 (Nsp1) inhibits the innate immune response by inserting its C-terminal helices into the mRNA entry channel of the ribosome and promoting mRNA degradation. Nevertheless, the mechanism by which Nsp1 achieves host translational shutoff while allowing for viral protein synthesis remains elusive. We set out to characterize the interactome of full-length Nsp1 and its topology by crosslinking mass spectrometry in order to investigate the role of the N-terminal domain and linker regions in host translational shutoff. We find that these regions are in contact with 40S proteins lining the mRNA entry channel and detect a novel interaction with the G subunit of the eIF3 complex. The crosslink-derived distance restraints allowed us to derive an integrative model of full-length Nsp1 on the 40S subunit, reporting on the dynamic interface between Nsp1, the ribosome and the eIF3 complex. The significance of the Nsp1-eIF3G interaction is supported by further evidence that Nsp1 predominantly binds to 40-43S complexes. Our results point towards a mechanism by which Nsp1 is preferentially recruited to canonical initiation complexes, leading to selective inhibition of host-translating ribosomes and subsequent mRNA degradation.

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Vibrational energy transfer (VET) in proteins has been linked to allostery and other aspects of protein function in computational studies. In their Communication on page 2899 ff., J. Bredenbeck, N. Budisa, and co‐workers show how vibrational energy can be injected and tracked experimentally in a site‐selective way. By using a genetically encoded ultrafast heater and femtosecond IR spectroscopy, this approach allowed them to map out VET pathways in proteins and to explore the role of VET for protein function.