Ortsaufgelöste Beobachtung von Schwingungsenergietransfer durch ein genetisch codiertes ultraschnelles Heizelement

Abstract

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.

Site-Specific 1D and 2D IR Spectroscopy to Characterize the Conformations and Dynamics of Protein Molecular Recognition

“…Along these lines, a recent study showed the possibility to measure vibrational energy transfer between multiple unnatural azido-labeled amino acids selectively introduced in a PDZ domain and peptide ligand, demonstrating a potential method for determination of distances between sites within or between proteins. 148 While unnatural amino acids with vibrations that provide strong, frequency-resolved signals are currently the most promising candidates as 2D IR probes of proteins, our longterm aspiration is to utilize C−D bonds introduced at native amino acid residues to directly interrogate the protein and eliminate concerns about perturbation. At this time, application of C−D bonds as probes even via linear spectroscopy is challenging and methodological improvement is needed to facilitate widespread adoption.…”

“…Measurement of the coupling between two probes in different proteins is another possibility. Along these lines, a recent study showed the possibility to measure vibrational energy transfer between multiple unnatural azido-labeled amino acids selectively introduced in a PDZ domain and peptide ligand, demonstrating a potential method for determination of distances between sites within or between proteins …”

Sequence of Events during Peptide Unbinding from RNase S: A Complete Experimental Description

“…We attribute the fast process (<100 ps, peak I), which we cannot time-resolve with the current experimental setup (see the Methods section for details), to the actual isomerization of the azobenzene moiety and the heat it dissipated into its immediate environment. 31,32 For the subsequent process, we note that the photoswitch is covalently linked to the S-peptide; hence, it will be constrained immediately after isomerization, since the RNase S cannot follow instantaneously. The vibrational mode around 1480 cm −1 feels the strain of that linker.…”

Direct Enzymatic Synthesis of a Deep‐Blue Fluorescent Noncanonical Amino Acid from Azulene and Serine

“…To improve access to this useful ncAA, we have engineered a highly active enzyme catalyst that synthesizes enantiomerically pure AzAla from commercially available starting materials in a single step. Given that an engineered synthetase/tRNA pair has been reported for this ncAA, Tm Azul also closes the gap for in vivo synthesis and incorporation of AzAla …”

“…In contrast to Trp, its spectroscopic properties are insensitive to the environment, making it ideal in contexts in which local conditions and quenchers (e.g., Met, His) could complicate the analysis of fluorescent signals . Its qualities have been leveraged for FRET experiments to elucidate protein–protein interactions as well as vibrational energy transfer (VET) studies to probe anisotropic energy flow within proteins . Recently, a method for the synthesis of AzAla by Negishi cross‐coupling was described (Scheme A) .…”